Antisense compositions targeted to β1-adrenoceptor-specific mRNA and methods of use

ABSTRACT

Disclosed are antisense oligonucleotide, polynucleotide, and peptide nucleic acid compounds that specifically bind to mammalian mRNA encoding a β 1 -adrenoceptor polypeptide and that are useful in the control and/or treatment of cardiac dysfunction, hypertension, hypertrophy, myocardial ischemia, and other cardiovascular diseases in an affected mammal, and preferably, in a human subject. The antisense compounds disclosed herein, and pharmaceutical formulations thereof, provide sustained control of β 1 -adrenoceptor expression over prolonged periods, and achieve therapeutic effects from as little as a single dose. Administration of these antisense compositions to approved animal models resulted in a decrease in blood pressure, but no significant change in heart rate. Use of such antisense compositions in the reduction of β 1 -adrenoceptor polypeptides in a host cell expressing β 1 -adrenoceptor-specific mRNA, and in the preparation of medicaments for treating human and animal diseases, and in particular, hypertension and other cardiac dysfunction is also disclosed.

The present application is a continuation-in-part of U.S. patent application Ser. No. 09/152,717 filed Sep. 14, 1998, and issued Jul. 11, 2000 as U.S. Pat. No. 6,087,343, and PCT International patent application Serial No. PCT/US99/21007, filed, Sep. 14, 1999, and published Mar. 23, 2000 as WO 00/15783, the entire contents of each of which is specifically incorporated herein by reference in its entirety without disclaimer.

The United States government has certain rights in the present invention pursuant to Grant HL-27334 from the National Institutes of Health.

1.0 BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cardiovascular disease and hypertension. More particularly, it concerns antisense oligonucleotide compositions and methods that are useful for reducing hypertension, cardiac hypertrophy, and myocardial ischemia in animals, particularly in mammals such as humans. Specifically, the invention provides antisense oligonucleotide and polynucleotide compositions capable of binding to β₁-adrenoceptor (β₁-adrenergic receptor, β₁-AR)-specific mRNA and inhibiting translation of the β₁-adrenoceptor mRNA, thereby decreasing the number of β₁-AR polypeptides in cells capable of expressing this mRNA. Disclosed are antisense oligonucleotide and peptide nucleic acid compositions, pharmaceutical formulations thereof, and vectors encoding antisense oligonucleotides that specifically bind β₁-AR mRNA and alter expression of the mRNA in a host cell.

2. Description of Related Art

1.2.1 Hypertension

Hypertension is the result of increased arterial resistance to blood flow and left untreated can lead to various pathological consequences. Hypertension affects approximately 40 million people in the United States. Heart attack (Nicholls et al., 1998), kidney damage (Agodoa, 1998), stroke (Chamorro et al., 1998) and loss of vision (Satllworth and Waldron, 1997) are typical conditions that result from high blood pressure. When blood vessels are subjected to high pressure for extended periods of time, they respond by thickening, vasospasm, and internal build-up of lipids and plaques, a condition known as arteriosclerosis. Arteriosclerosis further causes a decreased blood flow to the kidneys, which respond by releasing the protease renin. An overactive renin-angiotensin system is often implicated in the development of hypertension and cardiovascular disease (Nicholls et al., 1998).

Hypertension is often called the “silent killer,” since half of the population afflicted with high blood pressure are unaware of the condition. Thus, an initial step in combating hypertension is early detection. Following diagnosis, actions must be taken to control the disorder.

1.2.2 Treatment of Hypertension

Currently, four major categories of hypertensive drugs are administered to treat high blood pressure: (1) Diuretics, typically the drug of choice when the abnormal blood pressure is not very high, increase the rate at which the body eliminates urine and salt, resulting in decreased blood pressure by reducing volume (Moser, 1998); (2) β-adrenergic blockers (β-blockers), typically prescribed in combination with diuretics, lower blood pressure and heart rate (Rodgers, 1998); (3) Calcium channel blockers work by preventing the entry of calcium into cells, which reduces vasoconstriction (Rosenthal, 1993); (4) angiotensin converting enzyme (ACE) inhibitors prevent the narrowing and constriction of blood vessels by blocking the production of the vasoconstrictive peptide angiotensin II (AT-II), a product of ACE (Rosenthal, 1993).

Unfortunately, the short lasting effects of these drugs often require multiple, even daily doses to be therapeutically effective. Poor compliance is a major problem with drug regimens and can lead to a hypertensive crisis if the drug is not taken as scheduled.

The sympathetic nervous system plays a crucial role in the regulation of blood pressure (BP), mainly through activating α- and β-adrenergic receptors β₁-ARs) in the effector organs, including heart, kidney, and blood vessels. Adrenergic-blocking agents, especially β-blockers, are commonly used in the treatment of hypertension, ischemic heart disease, and arrhythmia (reviewed in Sproat and Lopez, 1991). However, β-blockers cause several side effects, which are usually associated with their central nervous system (CNS) reaction (e.g., sleep disturbance, depression, impotence, dizziness and fatigue) and β₂-adrenergic antagonistic activity (e.g., increase in peripheral vascular resistance, worsening of asthma symptoms). In addition, because of their short half-life (3 to 10 hrs), β-blockers must be taken daily to be effective. Because a cardiovascular disease such as hypertension is a life-lone disorder, longer-lasting treatment without side effects would be desirable.

Although the precise mechanism underlying the antihypertensive effects of β-blockers remains unclear, it is generally accepted that they antagonize the β₁-AR activity in heart and kidney, decreasing cardiac output and plasma renin activity (Sproat and Lopez, 1991). A new approach to β₁-blockade has been designed that reduces the number of receptors. Antisense oligonucleotides (AS-ODN) or antisense DNA designed specifically against B₁-ARs might represent a new class of β-blockers. Antisense techniques, through a number of mechanisms (Phillips et al., 1997), can effectively downregulate the expression of target proteins. Clinical trials using antisense in targeting AIDS (Akhtar and Rossi, 1996), cancer (Dachs et al., 1997), and other genetic and acquired diseases (Yla, 1997) indicate their potential clinical usefulness. The antisense approach has several potential advantages over β-blockers. First, the specificity of AS-ODNs is based on DNA sequence. Second, AS-ODNs do not have direct CNS effects, because of the negligible transport of these highly polar molecules through the blood-brain barrier (Agrawal et al., 1991). Third, antisense elements tested in different systems produce long-term effects after single treatment (Gyurko et al., 1997). This prolonged effect can be attributed to 2 features of AS-ODN. One is the extended half-life of chemically modified ODN. The half-life 15-20-mer phosphorothioated ODN is 20 to 50 hrs in rats and mice after intravenous injection (Iversen, 1991; Zhang et al., 1995). The other is associated with the nature of antisense inhibition, which provides a delayed yet prolonged blockade of target proteins distinct from the direct competitive antagonists currently available.

1.2.3 β₁-Adrenoceptors

β₁-ARs, which are distributed in the heart, kidney and blood vessels, play a role in the physiological control of blood pressure. For many years β-blocker drugs have been used for the treatment of hypertension through a regimen of daily dosing. The mechanism of control of blood pressure is not precisely known but the value of beta-blockers in hypertension control has been underscored by the reports of the Joint National Committee on High Blood Pressure recommending β-blockers as the first line of defense in the treatment of hypertension. Current β-blocker drugs, however, have certain disadvantages, including: (1) they have to be taken daily, or twice a day and compliance is a problem; (2) they have central effects, leading to psychological changes that contribute to the problem of patient compliance; and (3) many of the β-blockers now available are not specific for β₁-ARs and, therefore, can have untoward side effects.

1.3 Deficiences in the Prior Art

As can be understood from the above, there remains a need for an effective β₁-AR blocker that is highly specific, nontoxic, produces few side effects and does not cross the blood brain barrier to produce psychological changes. Also, a β-blocker that would last several days or weeks would allow patients more flexibility in the regimen of drug dosage by taking drugs infrequently. Thus, the need exists for an effective treatment of cardiac deficiencies (including hypertension, hypertrophy, ischemias, and the like) that circumvents the toxic side effects of existing therapies and provides more specific β₁-AR inhibition with longer acting effects for improved patient compliance. In addition, methods for delivery of antisense oligonucleotides and polynucleotides to a host cell, and in particular, non-invasive administration of specific antisense constructs to a mammal such as a human subject is particularly desirable.

2.0 SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations in the prior art by providing antisense nucleic acid compounds and compositions comprising them that specifically inhibit or reduce the expression of an mRNA encoding a β₁-adrenoceptor (β₁-AR) polypeptide in a host cell expressing the mRNA. More specifically, the subject invention provides antisense oligonucleotides (AS-ONs) and antisense oligo-peptide nucleic acids (AS-PNAs) that can specifically bind to a mammalian β₁-AR mRNA, resulting in the reduction or inhibition of translation of the messenger ribonucleic acid (mRNA) into β₁-AR polypeptide. Such oligonucleotides and PNA may be readily formulated in pharmaceutically-acceptable vehicles and provided directly to host cells, or administered systemically to mammals that express β₁-AR mRNA to reduce the level of β₁-AR produced in such cells and affected mammals.

The invention also provides genetic constructs comprising substantially full-length, antisense polynucleotide (AS-PN) sequences operably linked to a suitable promoter that may be used to transform selected cells to endogenously express “antisense” mRNA sequences that are complementary to the native β₁-AR mRNA sequence. When expressed in a suitable host cell, these essentially full length anti-mRNAs specifically bind to the native β₁-AR mRNA produced in the same host cell, and effectively reduce the availability of native β₁-AR mRNA that can serve as a template for the translation machinery of the host cell to produce β₁-AR polypeptide.

The antisense compounds and the genetic constructs of the present invention may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects to inhibit or significantly reduce the expression of β₁-AR-specific mRNA, and/or to inhibit or significantly reduce the translation of β₁-AR-specific mRNA into functional β₁-AR polypeptide. The antisense compounds of the present invention, and compositions comprising them provide new and useful therapeutics for the treatment, control, and amelioration of symptoms of a variety of cardiovascular disorders including hypertension, ischemia, cardiac hypertrophy, myocardial infarction, cardiac dysfunction, and diseases of the heart, that result from, or are exacerbated by, expression of β₁-AR-specific mRNA in the host cells of the affected mammal. Moreover, pharmaceutical compositions comprising one or more of the nucleic acid compounds disclosed herein, provide significant advantages over existing conventional therapies—namely, (1) their reduced side effects, (2) their increased efficacy for prolonged periods of time, (3) their ability to increase patient compliance due to their ability to provide therapeutic effects following as little as a single dose of the composition to affected individuals.

Preferred antisense compounds for use in the practice of the present invention are those nucleic acid and peptide nucleic acid sequences that specifically bind to a gene or an mRNA encoding β₁-AR polypeptide and that inhibit the expression or reduce the level of β₁-AR polypeptide in a mammalian host cell that expresses the gene and/or the mRNA.

The compounds of the invention, and the pharmaceutical formulations thereof, permit the development of methods for treating hypertension, ischemia, cardiac hypertrophy, cardiac dysfunction, or other cardiovascular diseases through the administration of at least one antisense compound that specifically binds to a mammalian β₁-AR-specific gene or mRNA, wherein the binding of the antisense compound to the gene or the mRNA alters, decreases, inhibits, and/or prevents transcription of the β₁-AR-specific mRNA, or, alternatively alters, decreases, inhibits, and/or prevents translation of the β₁-AR-specific mRNA into functional β₁-AR polypeptide in a host cell. As described above, the present methods offer significant advantages over traditional pharmacological modalities involving drugs that block or interfere with activity of the β₁-AR polypeptide (and not the amount of β₁-AR polypeptide). The present methods also avoid many of the untoward side effects of conventional therapies, avoid invasive surgical procedures, and are effective in lower, less frequent dosing regimens. The ability to dose less frequently represents a key aspect of improving patient compliance in dosing and reduces administration costs associated with more frequent dosage regimens.

In a first embodiment, the invention provides antisense oligonucleotides that specifically bind to a mammalian β₁-AR-specific gene or a mammalian β₁-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, β₁-AR polypeptide produced in the cell.

In a second embodiment, the invention provides antisense peptide nucleic acids that specifically bind to a mammalian β₁-AR-specific gene or a mammalian β₁-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, β₁-AR polypeptide produced in the cell.

In a third embodiment, the invention provides antisense polynucleotides that specifically bind to a mammalian β₁-AR-specific gene or to a mammalian β₁-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, β₁-AR polypeptide produced in the cell. The antisense polynucleotides represent full-length, or substantially full-length sequences that are complementary to a mammalian β₁-AR-specific mRNA, and that when present in a cell that expresses a mammalian β₁-AR-specific mRNA, will specifically bind to the β₁-AR-specific mRNA, thereby reducing the availability of functional mRNA in the cell from which the cellular protein machinery can translate functional β₁-AR polypeptide. Preferably, these full-length, or substantially full-length polynucleotides are provided to a cell via a genetic construct that comprises a promoter capable of expressing the complementary mRNA in a selected host cell. Such genetic constructs may be provided to suitable host cells using any one of the conventional gene therapy modalities known to those of skill in the art, such as, for example by one or more of the viral, retroviral, adenoviral, or adenoassociated viral constructs commonly exploited for the delivery and expression of heterologous polynucleotides.

In a fourth embodiment, the invention provides compositions that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and a suitable diluent, carrier, or pharmaceutical vehicle.

In a fifth embodiment, the invention provides pharmaceutical formulations that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and at least one antihypertensive pharmaceutical compound.

In a sixth embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds.

In a seventh embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds in combination with at least one antihypertensive pharmaceutical compound.

In an eighth embodiment, the invention provides therapeutic kits and medicaments that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the treatment of mammalian cardiac dysfunction or disease. Alternatively, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the preparation of genetic constructs for the development of suitable gene therapy vectors. Likewise, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the formulation of multi-drug “cocktails” for the treatment of one or more pathological conditions.

The invention also provides methods for the treatment of one or more cardiac diseases or dysfunctions employing one or more of the antisense compounds or compositions as described herein.

In each of the embodiments described herein, the oligonucleotide, polynucleotide, or peptide nucleic acid compound comprises a sequence region least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian β₁-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity of β₁-AR in a host cell expressing the mRNA.

2.1 Oligonucleotide and Oligo-PNA Compounds

In oligonucleotide and oligo-peptide nucleic acid embodiments, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least nine, at least ten, at least eleven, at least twelve, at least thirteen at least fourteen, or at least fifteen contiguous bases from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, as well as the smaller n-mer sequences illustrated in SEQ ID NO:29 through SEQ ID NO:186.

Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, or at least twenty-five contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, or at least thirty-four contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

Alternatively, the oligonucleotide or PNA may consist of a sequence of from about nine to about 35 nucleotides in length, and may comprise the entire sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

The oligonucleotide or PNA compound may consist essentially of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.

The oligonucleotide or PNA compound may also consist of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.

As described above, the length of the preferred oligonucleotide or PNA compounds preferably will be on the order of from about nine to about 35 or so nucleotides. As such, in addition to those compounds that are nine nucleotides in length, and those compounds that are 35 nucleotides in length, all intermediate size compounds are also contemplated to be useful in the practice of the present invention. Thus, oligonucleotide or PNA compounds that are ten nucleotides in length, eleven nucleotides in length, twelve nucleotides in length, thirteen nucleotides in length, fourteen nucleotides in length, fifteen nucleotides in length, sixteen nucleotides in length, seventeen nucleotides in length, eighteen nucleotides in length, nineteen nucleotides in length, twenty nucleotides in length, twenty-one nucleotides in length, twenty-two nucleotides in length, twenty-three nucleotides in length, twenty-four nucleotides in length, twenty-five nucleotides in length, twenty-six nucleotides in length, twenty-seven nucleotides in length, twenty eight nucleotides in length, twenty-nine nucleotides in length, thirty nucleotides in length, thirty-one nucleotides in length, thirty-two nucleotides in length, thirty-three nucleotides in length, and thirty-four nucleotides in length are also contemplated to fall within the scope of the present disclosure. In fact, the preferred oligonucleotide or PNA compounds may also be slightly shorter than the preferred range, that is, they may be about six or about seven or about eight nucleotides in length, or they may even be slightly longer than the preferred range (i.e., they may be about thirty-six or about thirty-seven or even about thirty-eight or so nucleotides in length), and may still function to reduce the level of β₁-AR polypeptide in a cell, and thus, effective in the treatment of cardiac disorders resulting from an elevated level of β₁-AR polypeptide.

Likewise, there is no obligate requirement that the antisense compounds be exactly 100% complementary to a given target sequence on the mammalian β₁-AR mRNA. Nor is there an obligate requirement that the antisense compounds be exactly 100% identical to one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186. In fact, the only requirement is that the oligonucleotide or PNA compound have sufficient homology to the gene or the mRNA encoding β₁-AR polypeptide so that upon specifically binding to the complementary region, a reduction in either the transcription of the β₁-AR gene and/or a reduction in the translation of β₁-AR polypeptide from the mRNA is observed. Therefore, the compounds may have 4 or fewer, 3 or fewer, 2 or fewer, or even 1 mismatch from the target sequence to which it specifically binds, and may still be sufficiently active to cause a reduction in β₁-AR polypeptide within the host cell. Thus, in addition to those compounds disclosed above that comprise sequences that are 100% identical to the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186, the invention also encompasses antisense compounds that comprise sequence regions that are about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or even about 90% identical to a portion of one of those sequences, so long as the resulting degenerate nucleotide sequence retains sufficient homology so that it specifically binds to the gene or mRNA encoding β₁-AR polypeptide.

2.2 Polynucleotide and Poly-PNA Compounds

In certain embodiments, the polynucleotide comprises at least 9 contiguous bases from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In other aspects of the invention, the polynucleotide comprises DNA, RNA, PNA, or preferably a derivatized or modified polynucleotide, including phosphorothioate derivates and the like.

Preferably the antisense polynucleotide compound comprises a sequence that is substantially the full-length complement of the β₁-AR mRNA, wherein binding of the substantially full-length complementary sequence to the β₁-AR mRNA is effective in reducing the translation of the mRNA into the β₁-AR polypeptide. Preferably the substantially full-length complementary sequence is comprised within a suitable vector that may be delivered to appropriate host cells. The vector preferably comprises a nucleic acid segment that is operably linked to a promoter sequence that expresses the complementary antisense sequence in the host cell. This complementary near full-length sequence is then able to specifically bind to the substantially full length β₁-AR mRNA and thereby prevent or substantially reduce the translation of the mRNA into β₁-AR polypeptide. The term substantially full-length is meant to include those sequences that comprise a sequence that is complementary to a region that is at least about 80% or more of the native β₁-AR-specific mRNA. Thus, for a β₁-AR mRNA that is 1000 nucleotides in length, a suitable complementary substantially full-length complement is a sequence that is complementary to a region of at least 800 nucleotides of the particular β₁-AR mRNA. Likewise, all sequences that are greater than 80% of the full-length sequences (i.e. those that are at least 81% full length, at least 82% full length, at least 83% full length, at least 84% full length, at least lo 85% full length, at least 86% full length, at least 87% full length, at least 88% full length, at least 89% full length, at least 90% full length, at least 91% full length, at least 92% full length, at least 93% full length, at least 94% full length, at least 95% full length, at least 96% full length, at least 97% full length, at least 98% full length, and at least 99% full length are also contemplated to fall within the scope of the present disclosure.

2.3 Compositions Comprising a β₁-AR Antisense Compound and a Second Cardiac Therapeutic Agent

When desirable, the clinician may combine two or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed above to provide an antisense “cocktail” to effect a more substantial reduction in the levels of β₁-AR polypeptide. Likewise, the patient or animal to be treated may benefit from a combination therapy involving one or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed herein and at least one anti-hypertensive or other cardiac therapy medicament.

For example, the composition may compris at least a first anti-hypertensive agent. In particular embodiments, the anti-hypertensive agent is selected from the group consisting of captopril (Captopril®), enalapril (Vasotec®), ramipril (Altace®), fosinopril (Monopril®), lisinopril (Prinivil®, Zestril®), quinapril (Accupril®), benazepril (Lotensin®), trandolapril (Mavik®), and moexipril (Univasc®). In other embodiments, the anti-hypertensive agent is selected from the group of angiotensin receptor blockers consisting of candesartan (Atacand®), losartan (Cozaar® and Hizaar®), valsartan (Diovan®), and irbesartan (Avapro®).

In still other embodiments, the anti-hypertensive agent is selected from the group of diuretic consisting of dichlorphenamide (Daranide®), spironolactone (Aldactazide®), hydrochlorothiazide (Microzide® and Dyazide®), triamterene (Maxzide®), amiloride (Midamor® and Moduretic®), torsemide (Demadex®), ethacrynice acid (Edecrin®), furosemide (Lasix®), hydroflumethiazide (Diucardin®), chlorothiazide (Diuril®), methylclothiazide (Enduron®), polythiazide (Renese®), chlorthalidone (Thalitone®) and metolazone (Zaroxolyn®).

In still further embodiments, the anti-hypertensive agent is selected from the group of calcium channel blockers consisting of nifedepine (Adalat® and Procardia®), verapamil (Isoptin®, Verelan®, Calan® and Covera®), nicardipine (Cardene®), diltiazem (Tiazac®, Cardizem® and Dilacor®), isradipine (DynaCirc®), nimodipine (Nimotop®), amlodipine (Norvase®), felodipine (Plendil®), misoldipine (Sular®), and bepridil (Vasocor®).

In certain preferred embodiments, the composition may further comprise one or more pharmaceutically acceptable vehicles, exemplified by, but not limited to, liposomes, lipid particles, lipid vesicles, nanoparticles, microparticles, nanocapsules, nanospheres, and sphingosomes to facilitate administration of the antisense composition(s) to the affected patient or animal.

In certain aspects of the invention, the antisense composition of the present invention is specific for an mRNA encoding the human β₁-AR polypeptide. In particular embodiments, the host cell is a mammalian host cell. In certain preferred embodiments of the invention, the host cell is a human cell. In other preferred aspects, the host cell is comprised within a human.

2.4 Compositions Comprising a β₁-AR Antisense Compound and a Second Antisense Targeted to Another RAS Pathway Component

The present invention also provides compositions that comprise at least a first antisense oligonucleotide, PNA, or polynucleotide specific for a mammalian β₁-AR-specific mRNA as described above, and at least a second antisense compound specific for a mammalian renin, angiotensin, angiotensinogen, angiotensin type 1 (AT-1) receptor mRNA, or an ACE polypeptide.

In certain aspects of the invention, the second antisense compound is specific for a mammalian angiotensinogen mRNA, or an mRNA that encodes one or more RAS pathway-specific enzymes, such as a mammalian ACE polypeptide. In other aspects, the second antisense compound is specific for an mRNA that encodes a transcriptional factor.

Therapeutic combinations of three or more antisense compound are also provided. These cocktail therapies may comprise at least two antisense compounds specific for a mammalian β₁-AR-encoding gene or mRNA, and at least a third antisense compound specific for the same mRNA, or alternatively, an mRNA encoding another polypeptide in the RAS pathway as described above. The third antisense compound may be specific for renin, AT-1 receptor, ACE, or angiotensinogen, or another gene or mRNA involved in biochemical pathways involved in producing or regulating blood pressure and/or causing or contributing to hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in an affected animal. Alternatively, the constructs may even be specific for one or more particular transcription factor(s) that regulate one or more genes involved in producing hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in a mammal. Such combined therapy approached using two or more antisense oligonucleotides are particularly desirable where enhanced or synergistic activity towards treating hypertension ischemia, cardiac hypertrophy, or other cardiac dysfunction is achieved.

The invention further provides a method for reducing expression of a gene encoding manmmalian β₁-AR polypeptide in a host cell, the method comprising providing to the host cell an amount of an antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to an mRNA encoding the β₁-AR polypeptide, effective to reduce the amount of β₁-AR polypeptide in the cell.

Additionally, the invention provides a method for reducing the number of mammalian β₁-AR polypeptides in a cell, the method comprising introducing into the cell at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to all, substantially-all, or a portion of the mRNA expressed from a gene encoding a mammalian β₁-adrenoceptor polypeptide, and further wherein binding of the first antisense oligonucleotide, polynucleotide, or peptide nucleic acid to the mRNA is effective in reducing the number of mammalian β₁-AR polypeptides in the host cell expressing the mRNA.

The invention also provides a method for decreasing or treating hypertension in an animal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian β₁-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the number of such polypeptides in a host cell expressing the mRNA.

The invention additionally provides a method for treating a disease associated with elevated β₁-AR activity in a mammal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian β₁-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the receptor activity or receptor number in a host cell expressing the mRNA, such that a decrease in β₁-adrenoceptor activity is effected, thereby resulting in amelioration of the disease that results from, or is exacerbated by, an elevated level of β₁-AR activity in the affected animal.

The invention also provides a method for treating a cardiac hypertrophic or ischemic disease that is associated with, that results from, or is exacerbated by, the presence of, or an elevation in, β₁-adrenoceptor polypeptides in the affected animal. This method generally involves the administration to the animal of one or more compositions that comprise at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian β₁-AR polypeptide in an amount, and for a time sufficient to decrease the number, amount, or activity of the β₁-adrenoceptor polypeptide in a host cell expressing the mRNA.

Further provides are kits for treating hypertension in a human comprising: (a) a pharmaceutically-acceptable formulation comprising at least a first oligonucleotide of at least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian β₁-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity, amount, or number of receptor polypeptides in a host cell expressing the mRNA; a pharmaceutical excipient; and (b) instructions for using the kit.

In addition to methods involving the delivery of exogenous oligonucleotide compositions to a host cell, or administration of such compositions to an animal in a therapeutic pharmaceutical formulation, the present invention also concerns gene therapy methods for introducing into a host cell a DNA construct that is transcribed by the cell machinery to give rise to an antisense RNA molecule that specifically binds to a portion of an mRNA encoding a mammalian β₁-AR polypeptide. In a preferred embodiment, such therapies may be accomplished through the use of viral vectors, such as retro-, adeno- or adeno-associated viruses as described hereinbelow.

Regulation of expression of a gene encoding a mammalian β₁-AR polypeptide in a mammalian cell genomes may also be achieved by integration of a gene under the transcription control of a promoter which is functional in the host and in which the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous β₁-AR-specific polynucleotide sequence(s) one wishes to regulate. The integrated gene, referred to as an antisense gene, provides an RNA sequence capable of binding to naturally existing RNAs, exemplified by mammalian β₁-AR polypeptide-specific mRNA, and inhibiting its expression, where the anti-sense sequence may bind to the coding, non-coding, or both, portions of the RNA. The antisense construction may be introduced into the animal cell in a variety of ways and be integrated into the animal genome for inducible or constitutive transcription of the antisense sequence.

Another aspect of the invention provides a pharmaceutical composition useful for inhibiting expression of mammalian β₁-AR-specific mRNA comprising a pharmaceutical carrier and one or more antisense oligonucleotides of the present invention that specifically bind to and reduce expression of the specific mRNA. Another aspect of the invention provides a method for treating hypertension in a human comprising administering to a subject an effective amount of at least one oligonucleotide composition as described herein, in an amount effective to reduce hypertension in the human, or to ameliorate the degree or extent of hypertension in the patient.

2.5 Definitions

The term “β₁-AR” refers to polypeptides having amino acid sequences which are substantially similar to the native mammalian β₁-AR polypeptide amino acid sequences and which are biologically active and/or which cross-react with β₁-AR polypeptide-specific antibodies raised against a mammalian β₁-AR polypeptide or peptide fragment thereof.

The term “β₁-AR” also includes analogs of mammalian β₁-AR polypeptides that exhibit at least some biological activity in common with native mammalian β₁-AR polypeptides.

Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct mammalian β₁-AR polypeptide analogs or β₁-AR fusion proteins or identify β₁-AR-related mRNAs and/or genes using well-known molecular biology techniques, including those described herein. Oligonucleotides complementary to mammalian β₁-AR polypeptide-encoding mRNAs form the heart of the present invention, especially human β₁-AR polypeptide-encoding mRNAs.

The oligonucleotides (or “ODNs” or “polynucleotides” or “oligos” or “oligomers” or “n-mers”) of the present invention are preferably deoxyoligonucleotides (ie. DNAs), or derivatives thereof; ribo-oligonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.

The term “substantially complementary,” when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an Mrna encoding a mammalian β₁-AR polypeptide. As such, typically the sequences will be highly complementary to the Mrna “target” sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the Mrna and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target Mrna sequence into polypeptide product.

Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or ‘% exact-match’) to the corresponding Nrna target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary oligonucleotide sequences for use in the practice of the invention, and in such instances, the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to all or a portion of the target Mrna to which the designed oligonucleotide specifically binds.

Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

2.6 Exemplary Antisense Constructs for β₁-Adrenoceptor-Specefic mRNA

Table 1 lists representative oligonucleotide sequences contemplated for use in the practice of the present invention.

TABLE 1 EXEMPLARY OLIGONUCLEOTIDE SEQUENCES TARGETED TO MAMMALIAN β₁-AR-ENCODING MRNAS SEQ ID NO: SEQUENCE Sequences homologous to rat β₁-AR mRNA SEQ ID NO:1 5′-CCGCGCCCATGCCGA-3′ SEQ ID NO:2 5′-GGCCGACGACAGGTT-3′ SEQ ID NO:3 5′-ATGAGCAGCACGATG-3′ SEQ ID NO:4 5′-GGGCGCTCGCCCTGGCGCCTCCGAACCCTGCAACC-3′ SEQ ID NO:5 5′-ATGGGCGCGGGGGCGCTCGCCCTGGCGCCTCCGAA-3′ SEQ ID NO:6 5′-GCCTCCGAATCGGCATGGGCGCGGGGGCGCTCGCC-3′ SEQ ID NO:7 5′-ACCCCCCGCGCCCGGCCTCCGAATCGGCATGGGCG-3′ Sequences homolagous to human 131-AR mRNA SEQ ID NO:8 5′-GGGTGCTCGTCCTGGGCGCCTCCGAGCCCGGTAAC-3′ SEQ ID NO:9 5′-ATGGGCGCGGGGGTGCTCGTCCTGGGCGCCTCCGA-3′ SEQ ID NO:10 5′-GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG-3′ SEQ ID NO:11 5′-CCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGTG-3′ SEQ ID ND:12 5′-CCGCCCCCGGCCTCCGCAGCTCGGCATGGGCGCGG-3′ SEQ ID NO:13 5′-ACCCCCCGCCCCCGGCCTCCGCAGCTCGGCATGGG-3′ Sequences homologous to murine β₁-AR mRNA SEQ ID NO:14 5′-GGGCGCTCGCCCTGGGCGCCTCCGAACCCTGCAAC-3′ SEQ ID NO:15 5′-ATGGGCGCGGGGGCGCTCGCCCTGGGCGCCTCCGA-3′ SEQ ID NO:16 5′-GCAGCTCGGCATGGGCGCGGGGGCGCTCGCCCTGG-3′ SEQ ID NO:17 5′-CCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGCG-3′ Sequences homalogous to sheep β₁-AR mRNA SEQ ID NO:18 5′-GGGCGCTCGCCCTGGGCGCCTCCGAGCCCTGCAAC-3′ SEQ ID NO:19 5′-ATGGGCGCGGGGGCGCTCGCCCTGGGCGCCTCCGA-3′ SEQ IO NO:20 5′-GCAGCTCGGCATGGGCGCGGGGGCGCTCGCCCTGG-3′ SEQ ID NQ:21 5′-CCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGCG-3′ Sequences homalogous to porcine β₁-AR mRNA SEQ ID NO:22 5′-GCGGGGGCGTCGCCCTGGGTGCCTCCGAGCCCTGC-3′ SEQ ID NO:23 5′-ATGGGGCGGGGGCGTCGCCCTGGGTGCCTCCGAGC-3′ SEQ ID NO:24 5′-CGCAGCCGGTATGGGGCGGGGGCGTCGCCCTGGGT-3′ SEO ID NO:25 5′-CCCCCGCCTCCGCA6CCGGTAT6GG6C66GG6CGT-3′ Sequences homologous to monkey β₁-AR mRNA SEO ID NO:26 5′-GGGCGCTCGTCCTGGGCGCCTCCGA6CCC6GTAAC-3′ SEQ ID ND:27 5′-ATGGGCGCGGGGGCGCTCGTCCTGGGCGCCTCCGA-3′ SEQ ID ND:28 5#-GCAACTCGGCATGGGCGCGGGG6CGCTCGTCCTGG-3′

N means any nucleotide, e.g., C, A, U (T), or G.

Sequences in bold correspond to portions of the β₁-AR open reading frame.

In addition to the indicated 35-mers, smaller internal n-mers that comprise from at least 9 bases in length up to the full length 35-mers listed are also contemplated to be useful in the practice of the present invention. For example, in addition to the first indicated full-length oligomer, all internal n-mers are also considered to fall within the scope of this disclosure. Thus for each of the 35-mers (SEQ ID NO:4 to SEQ ID NO:28) all internal 34-mers of each sequence as well as all internal 33-mers, 32-mers, 31-mers, 30-mers, 29-mers, 28-mers, 27-mers, 26-mers, 25-mers, 24-mers, 23-mers, 22-mers, 21-mers, 20-mers, 19-mers, 18-mers, 17-mers, 16-mers, 15-mers, 14-mers, 13-mers, 12-mers, 11-mers, 10-mers, and 9-mers, that are comprised within of each of the disclosed 35-mers are also contemplated to be useful in the practice of the present invention.

For illustrative purposes, all representative n-mers of SEQ ID NO:10 would include the following internal contiguous 9-mer to 34-mer sequences:

CAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:29)

GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTG (SEQ ID NO:30)

AGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:31)

GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCT (SEQ ID NO:32)

GCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:33)

GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCC (SEQ ID NO:34)

CTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:35)

GCAGCTCGGCATGGGCGCGGGGGTGCTCGTC (SEQ ID NO:36)

TCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:37)

GCAGCTCGGCATGGGCGCGGGGGTGCTCGT (SEQ ID NO:38)

CGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:39)

GCAGCTCGGCATGGGCGCGGGGGTGCTCG (SEQ ID NO:40)

GGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:41)

GCAGCTCGGCATGGGCGCGGGGGTGCTC (SEQ ID NO:42)

GCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:43)

GCAGCTCGGCATGGGCGCGGGGGTGCT (SEQ ID NO:44)

CATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:45)

GCAGCTCGGCATGGGCGCGGGGGTGC (SEQ ID NO:46)

ATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:47)

GCAGCTCGGCATGGGCGCGGGGGTG (SEQ ID NO:48)

TGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:49)

GCAGCTCGGCATGGGCGCGGGGGT (SEQ ID NO:50)

GGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:51)

GCAGCTCGGCATGGGCGCGGGGG (SEQ ID NO:52)

GGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:53)

GCAGCTCGGCATGGGCGCGGGG (SEQ ID NO:54)

GCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:55)

GCAGCTCGGCATGGGCGCGGG (SEQ ID NO:56)

CGCGGGGGTGCTCGTCCTGG (SEQ ID NO:57)

GCAGCTCGGCATGGGCGCGG (SEQ ID NO:58)

GCGGGGGTGCTCGTCCTGG (SEQ ID NO:59)

GCAGCTCGGCATGGGCGCG (SEQ ID NO:60)

CGGGGGTGCTCGTCCTGG (SEQ ID NO:61)

GCAGCTCGGCATGGGCGC (SEQ ID NO:62)

GGGGGTGCTCGTCCTGG (SEQ ID NO:63)

GCAGCTCGGCATGGGCG (SEQ ID NO:64)

GGGGTGCTCGTCCTGG (SEQ ID NO:65)

GCAGCTCGGCATGGGC (SEQ ID NO:66)

GGGTGCTCGTCCTGG (SEQ ID NO:67)

GCAGCTCGGCATGGG (SEQ ID NO:68)

GGTGCTCGTCCTGG (SEQ ID NO:69)

GCAGCTCGGCATGG (SEQ ID NO:70)

GGTGCTCGTCCTGG (SEQ ID NO:71)

GCAGCTCGGCATG (SEQ ID NO:72)

GTGCTCGTCCTGG (SEQ ID NO:73

GCAGCTCGGCAT (SEQ ID NO:74)

TGCTCGTCCTGG (SEQ ID NO:75)

GCAGCTCGGCA (SEQ ID NO:76)

GCTCGTCCTGG (SEQ ID NO:77)

GCAGCTCGGC (SEQ ID NO:78)

CTCGTCCTGG (SEQ ID NO:79)

GCAGCTCGG (SEQ ID NO:80), Etc.

In similar fashion, for illustrative purposes, and in the sake of brevity, representative n-mers of SEQ ID NO:1 would include the following internal contiguous sequences:

CGCGCCCATGCCGA (SEQ ID NO:81);

CCGCGCCCATGCCG (SEQ ID NO:82);

GCGCCCATGCCGA (SEQ ID NO:83);

CCGCGCCCATGCC (SEQ ID NO:84);

CGCCCATGCCGA (SEQ ID NO:85);

CCGCGCCCATGC (SEQ ID NO:86);

GCCCATGCCGA (SEQ ID NO:87);

CCGCGCCCATG (SEQ ID NO:88);

CCCATGCCGA (SEQ ID NO:89);

CCGCGCCCAT (SEQ ID NO:90);

CCATGCCGA (SEQ ID NO:91); and

CCGCGCCCA (SEQ ID NO:92).

Given the benefit of the present disclosure, the skilled artisan would also be able now, in similar fashion, to prepare any and all possible n-mers from each of the disclosed sequences, and from these sequences, identify and select those particular oligonucleotide sequences that specifically inhibit β₁-AR-specific mRNA expression for therapeutic use by using an acceptable in vitro and/or in vivo assay, such as those described hereinbelow.

Likewise, based upon the sequence of the human β₁-AR gene, the inventors have identified highly preferred sequences of from about 15 to about 25 nucleotides in length that specifically bind to the mRNA encoding β₁-AR polypeptide, and that reduce the level of polypeptide in a cell expressing such an mRNA. Illustrative sequences in this size range are identified below:

Length=15 nucleotides:

5′-CACCCCCGCGCCCAT-3′ (SEQ ID NO:93)

5′-ACCCCCGCGCCCATG-3′ (SEQ ID NO:94)

5′-CGCGCCCATGCCGAG-3′ (SEQ ID NO:95)

5′-GCGCCCATGCCGAGC-3′ (SEQ ID NO:96)

5′-CGCCCATGCCGAGCT-3′ (SEQ ID NO:97)

5′-GCCCATGCCGAGCTG-3′ (SEQ ID NO:98)

5′-CCCATGCCGAGCTGC-3′ (SEQ ID NO:99)

5′-CCATGCCGAGCTGCG-3′ (SEQ ID NO:100)

5′-CATGCCGAGCTGCGG-3′ (SEQ ID NO:101)

5′-ATGCCGAGCTGCGGA-3′ (SEQ ID NO:102)

Length=16 nucleotides:

5′-GCGCCCATGCCGAGCT-3′ (SEQ ID NO:103)

5′-CGCCCATGCCGAGCTG-3′ (SEQ ID NO:104)

5′-GCCCATGCCGAGCTGC-3′ (SEQ ID NO:105)

5′-CCCATGCCGAGCTGCG-3′ (SEQ ID NO:106)

5′-CCATGCCGAGCTGCGG-3′ (SEQ ID NO:107)

5′-CATGCCGAGCTGCGGA-3′ (SEQ ID NO:108)

5′-ATGCCGAGCTGCGGAG-3′ (SEQ ID NO:109)

5′-TGCCGAGCTGCGGAGG-3′ (SEQ ID NO:110)

Length=17 nucleotides:

5′-CGCGCCCATGCCGAGCT-3′ (SEQ ID NO:111)

5′-GCGCCCATGCCGAGCTG-3′ (SEQ ID NO:112)

5′-CGCCCATGCCGAGCTGC-3′ (SEQ ID NO:113)

5′-GCCCATGCCGAGCTGCG-3′ (SEQ ID NO:114)

5′-CCCATGCCGAGCTGCGG-3′ (SEQ ID NO:115)

5′-CCATGCCGAGCTGCGGA-3′ (SEQ ID NO:116)

5′-CATGCCGAGCTGCGGAG-3′ (SEQ ID NO:117)

5′-ATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:118)

Length=18 nucleotides:

5′-CCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:119)

5′-CGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:120)

5′-GCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:121)

5′-CGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:122)

5′-GCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:123)

5′-CCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:124)

5′-CCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:125)

5′-CATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:126)

Length=19 nucleotides:

5′-ACCCCCGCGCCCATGCCGA-3′ (SEQ ID NO:127)

5′-CCCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:128)

5′-CCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:129)

5′-CGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:130)

5′-GCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:131)

5′-CGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:132)

5′-GCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:133)

5′-CCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:134)

5′-CCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:135)

Length=20 nucleotides:

5′-CACCCCCGCGCCCATGCCGA-3′ (SEQ ID NO:136)

5′-ACCCCCGCGCCCATGCCGAG-3′ (SEQ ID NO:137)

5′-CCCCCGCGCCCATGCCGAGC-3′ (SEQ ID NO:138)

5′-CCCCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:139)

5′-CCCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:140)

5′-CCGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:141)

5′-CGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:142)

5′-GCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:143)

5′-CGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:144)

5′-GCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:145)

5′-CCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:146)

Length=21 nucleotides:

5′-CACCCCCGCGCCCATGCCGAG-3′ (SEQ ID NO:147)

5′-ACCCCCGCGCCCATGCCGAGC-3′ (SEQ ID NO:148)

5′-CCCCCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:149)

5′-CCCCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:150)

5′-CCCGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:151)

5′-CCGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:152)

5′-CGCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:153)

5′-GCGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:154)

5′-CGCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:155)

5′-GCCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:156)

Length=22 nucleotides:

5′-CACCCCCGCGCCCATGCCGAGC-3′ (SEQ ID NO:157)

5′-ACCCCCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:158)

5′-CCCCCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:159)

5′-CCCCGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:160)

5′-CCCGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:161)

5′-CCGCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:162)

5′-CGCGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:163)

5′-GCGCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:164)

5′-CGCCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:165)

Length=23 nucleotides:

5′-CACCCCCGCGCCCATGCCGAGCT-3′ (SEQ ID NO:166)

5′-ACCCCCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:167)

5′-CCCCCGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:168)

5′-CCCCGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:169)

5′-CCCGCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:170)

5′-CCGCGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:171)

5′-CGCGCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:172)

5′-GCGCCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:173)

Length=24 nucleotides:

5′-CACCCCCGCGCCCATGCCGAGCTG-3′ (SEQ ID NO:174)

5′-ACCCCCGCGCCCATGCCGAGCTGC-3′ (SEQ ID NO:175)

5′-CCCCCGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:176)

5′-CCCCGCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:177)

5′-CCCGCGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:178)

5′-CCGCGCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:179)

5′-CGCGCCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:180)

Length=25 nucleotides:

5′-CACCCCCCGCGCCATGCCGAGCTGC-3′ (SEQ ID NO:181)

5′-ACCCCCGCGCCCATGCCGAGCTGCG-3′ (SEQ ID NO:182)

5′-CCCCCGCGCCCATGCCGAGCTGCGG-3′ (SEQ ID NO:183)

5′-CCCCGCGCCCATGCCGAGCTGCGGA-3′ (SEQ ID NO:184)

5′-CCCGCGCCCATGCCGAGCTGCGGAG-3′ (SEQ ID NO:185)

5′-CCGCGCCCATGCCGAGCTGCGGAGG-3′ (SEQ ID NO:186)

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A shows β₁-AS-ODN decreased density of β₁-ARs but not β₂-ARs in cardiac ventricles of SHRs. A¹ Time Course of β₁-AS-ODN effects on B_(max) of β₁-ARs () and β₂-ARs (∘).

FIG. 1B shows B_(max) of β-ARs 4 days after intravenous injection of saline (solid bar), 1 mg/kg inverted ODN (open bar), or 1 mg/kg β₁-AS-ODN (shaded bar). Data represent mean±SEM of each point (n=6). *P<0.01, **P<0.001 vs. saline control.

FIG. 2A and FIG. 2B show β₁-AS-ODN reduced ISO-stimulated positive inotropic more than chronotropic response in isolated perfused SHR hearts. 48 hr after intravenous injection of 1 mg/kg inverted ODN (∘) or β₁-AS-ODN (), hearts were perfused with Krebs buffer containing increasing concentrations of ISO.

FIG. 2A shows the effect of β₁-AS-ODN on ISO-induced elevation in left ventricular pressure. Data represent mean±SEM of each group (n=6 to 9). *P<0.05, **P<0.01 vs. inverted ODN.

FIG. 2B shows the effect of β₁-AS-ODN on ISO-induced elevation in HR.

FIG. 3A, FIG. 3B and FIG. 3C show in vivo effects of β₁-AS-ODN and atenolol on cardiovascular hemodynamics of SHRs in response to β₁-stimulation. Same group of SHRs (n=4) was tested for dobutamine-induced hemodynamic alteration at control levels (∘), and 1 hr. after atenolol injection (▴). Rats were allowed to fully recover between treatments. During dobutamine infusion,

FIG. 3A shows cardiac dP/dt_(max) of SHRs as monitored by the telemetry system. Data represent mean±SEM. *P<0.01 vs. control.

FIG. 3B shows HR of SHRs as monitored by telemetry system. Data represent mean±SEM. *P<0.01 vs. control.

FIG. 3C shows SBP of SHRs as monitored by telemetry system. Data represent mean±SEM. *P<0.01 vs. control.

FIG. 4 shows the effect of β₁-AS-ODN on SBP of SHRs measured with tail cuff. A single dose of 1 mg/kg β₁-AS-ODN was injected into tongue vein with cationic liposome at molar ratio of 1:05 (▪) or 1:2.5 (). Inverted ODN (1 mg/kg) delivered with liposomes at molar ratio of 1:2.5 (∘) served as control. Data represent mean±SEM of each group (n=6). *P<0.05 vs. inverted ODN.

FIG. 5A shows the effect of β₁-AS-ODN on mean BP of SHRs monitored by telemetry. Dose of 1 mg/kg inverted ODN (∘) or β₁-AS-ODN () was injected with liposomes at molar ratio of 1:05. BP and HR were recorded every 10 min. and averaged every 24 hr. Data represent mean±SEM of each group (n=6). *P<0.05 vs. inverted ODN.

FIG. 5B show the effect of β₁-AS-ODN on mean HR of SHRs monitored by telemetry. Dose of 1 mg/kg inverted ODN (∘) or β₁-AS-ODN () was injected with liposomes at molar ratio of 1:05. BP and HR were recorded every 10 min. and averaged every 24 hr. Data represent mean±SEM of each group (n=6). *P<0.05 vs. inverted ODN.

FIG. 6A shows the effect of atenolol on mean BP of SHRs monitored by telemetry. Saline (∘) or 1 mg/kg atenolol () was injected intravenously. BP and HR were taken every 10 min. and averaged every 1 hr. Data represent mean±SEM of each group (n=6). *P<0.05 vs. saline.

FIG. 6B shows the effect of atenolol on mean HR of SHRs monitored by telemetry. Saline (∘) or 1 mg/kg atenolol () was injected intravenously. BP and HR were taken every 10 min. and averaged every 1 hr. Data represent mean±SEM of each group (n=6). *P<0.05 vs. saline.

FIG. 7 shows improving the antihypertensive effect of β₁-AS-ODN by optimization of liposome:ODN charge ratios. SHR received a single intravenous injection of β₁-AS-ODN or inverted ODN. β₁-AS-ODN 0.5 mg/kg was delivered by DOTAP/DOPE at charge ratios from 0 to 3.5. Inverted ODN 0.5 mg/kg delivered by DOTAP/DOPE at charge ratio 2.0 served as control. Data represent mean values of each group (n=6). Standard errors were omitted for clarity.

FIG. 8A, FIG. 8B and FIG. 8C show the effects of β₁-AS-ODN on blood pressure and β-AR levels in renal cortex. SHR were injected with 0.5 mg/kg β₁-AS-ODN or inverted ODN with liposomes at charge ratio 2.

FIG. 8A shows the effect on blood pressure.

FIG. 8B shows the time course of the changes in B_(max) of β₁-AR () and β₂-AR (◯).

FIG. 8C shows the B_(max) of β-AR 4 days after intravenous injection of saline (solid bar), inverted ODN (open bar), or β₁-AS-ODN (shaded bar). Data represent mean±SEM of each point (n=6). *P<0.101 vs. saline control.

FIG. 9A, FIG. 9B and FIG. 9C show β₁-AS-ODN at a single injection exerts a delayed suppression on RAS.

FIG. 9A shows the effect on preprorenin mRNA levels in renal cortex.

FIG. 9B shows the effect on PRA.

FIG. 9C shows the effect on plasma Ang II levels. Data represent mean±SEM of each point (n=6). *P<0.01, **P<0.001 vs. control.

FIG. 10 illustrates sustained antihypertensive effects of repeated administrations of β₁-AS-ODN in SHR. Three repeated i.v. injections of 1 mg/kg β₁-AS-ODN (n=9) delivered by liposomes were given to adult SHR as indicated by upward arrows. Repeated injection of saline (n=7) and 1 mg/kg INV-ODN (n=7) served as controls. Systolic BP was measured by tail cuff method. Blood samples were collected at different time points as indicated by downward arrows for safety profile analysis. Data represent mean±SEM. P<0.05 vs. saline or INV-ODN.

FIG. 11 illustrates repeated administration of β₁-AS-ODN causes reduced left ventricular hypertrophy in SHR. SHR were treated with repeated injections of β₁-AS-ODN (n=9), saline (n=7) or INV-ODN (n=7) for 2 months. *P<0.

FIG. 12 illustrates β₁-AS-ODN and atenolol attenuated ischemia-reperfusion induced cardiac dysfunction. Note the marked increase in LVEDP and CPP and decrease in dLVP in saline-treated rat hearts exposed to a brief ischemia-reperfusion. β₁-AS-ODN, but not INV-ODN, exhibited beneficial effects on cardiac functions, indicated by a smaller increment in CPP and LVEDP and preservation dLVP. Atenolol showed a similar protective effect. Data represent mean±SEM of n=7 for each group.

FIG. 13 illustrates plasma levels of liver transaminases ALT and AST after repeated injections of β₁-AS-ODN. Saline and INV-ODN serve as controls. Data represent mean±SEM of each group (n=7-9).

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 4.1 Antisense Oligonucleotides

The utility of the disclosed compounds and compositions in inhibiting expression of mammalian β₁-AR-specific mRNA has been described and demonstrated both in vitro and in vivo by the methods described herein.

Preferred polynucleotide and oligonucleotide compounds of the present invention specifically bind to an mRNA encoding a mammalian β₁-AR polypeptide thereby inhibiting the translation of the mRNA, and concomitant expression of the encoded polypeptide.

Highly preferred antisense compounds and compositions are those that specifically bind to the mRNA encoding human β₁-AR polypeptide.

The mRNA sequence encoding the human β₁-AR polypeptide is disclosed in GenBank™ accession number NM000684, and described herein as SEQ ID NO:187. The mRNA sequence encoding canine β₁-AR polypeptide is disclosed in GenBank™ accession number U73207, and described herein as SEQ ID NO:188. The mRNA sequence encoding the sheep β₁-AR polypeptide is disclosed in GenBank™ accession numbers S78499 and AF072433, and described herein as SEQ ID NO:189. Likewise, porcine β₁-AR-encoding mRNA is disclosed in GenBank™ accession number AF042454, and described herein as SEQ ID NO:190. Rodent (Rattus) β₁-AR-encoding mRNA is disclosed in GenBank™ accession number D00634 and described herein as SEQ ID NO:191. Rhesus monkey β₁-AR-encoding mRNA is disclosed in GenBank™ accession number X75540, and described herein as SEQ ID NO:192. Murine (Mus) mouse β₁-AR-encoding mRNA is disclosed in GenBank™ accession number L10084, and described herein as SEQ ID NO:193. Highly homologous β₁-AR-encoding DNAs have also been identified in non-mammalian species, such as frog (SEQ ID NO:194) disclosed in GenBank™ as accession number Y09213, further evidence to the fact that antisense oligonucleotides prepared complementary to a β₁-AR-encoding DNA from one species may be useful in reducing the expression of β₁-AR-specific mRNA in another species. In fact, the inventors contemplate that antisense oligonucleotides prepared complementary to highly conserved regions of the proximal portion of the β₁-AR mRNA may specifically bind to, and reduce translation of, the mRNA in a variety of host cells and animals that produce β₁-adrenoceptor polypeptides.

In the specification and claims, the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura). As used in the specification and claims, compounds that are antisense to the β₁-adrenoceptor-specific PNA, DNA or mRNA sense strand are compounds that have a nucleoside sequence complementary to the sense strand. Table 2 shows the four possible sense strand nucleosides and their complements present in an antisense compound.

TABLE 2 Sense Antisense Ade Thy Gua Cyt Cyt Gua Thy Ade Ura Ade

It will be understood by those skilled in the art that the present invention broadly includes oligonucleotide compounds that are capable of binding to the DNA or mRNA sense strand coding for B₁R. It will also be understood that mRNA includes not only the ribonucleotide sequences encoding a protein, but also regions including the 5′-untranslated region, the 3′-untranslated region, the 5′-cap region and the intron/exon junction regions.

The invention includes compounds which are not strictly antisense; the compounds of the invention also include those oligonucleotides that may have some bases that are not complementary to bases in the sense strand provided such compounds have sufficient binding affinity for β₁-adrenoceptor DNA or mRNA to inhibit expression. In addition, base modifications or the use of universal bases such as inosine in the oligonucleotides of the invention are contemplated within the scope of the subject invention.

The antisense compounds may have some or all of the phosphates in the nucleotides replaced by phosphorothioates (X=S) or methylphosphonates (X=CH₃) or other C₁₋₄ alkylphosphonates. The antisense compounds optionally may be further differentiated from native DNA by replacing one or both of the free hydroxy groups of the antisense molecule with C₁₋₄ alkoxy groups (R=C₁₋₄ alkoxy). As used herein, C₁₋₄ alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon-atoms.

The disclosed antisense compounds also may be substituted at the 3′ and/or 5′ ends by a substituted acridine derivative. As used herein, “substituted acridine,” means any acridine derivative capable of intercalating nucleotide strands such as DNA. Preferred substituted acridines are 2-methoxy-6-chloro-9-pentylarninoacridine, N-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-3-aminopropanol, and N-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-5-aminopentanol. Other suitable acridine derivatives are readily apparent to persons skilled in the art. Additionally, as used herein “P(0)(0)-substituted acridine” means a phosphate covalently linked to a substitute acridine.

As used herein, the term “nucleotides” includes nucleotides in which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridines.

In one embodiment, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense ON. For example, the phosphates can be replaced by phosphorothioates. The ends of the molecule may also be optimally substituted by an acridine derivative that intercalates nucleotide strands of DNA. Intl. Pat. Appl. Publ. No. WO 98/13526 and U.S. Pat. No. 5,849,902 (each specifically incorporated herein by reference) describe a method of preparing three component chimeric antisense compositions, and discuss many of the currently available methodologies for synthesis of substituted oligonucleotides having improved antisense characteristics and/or half-life.

The reaction scheme involves ¹H-tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates that are subsequently reacted with sulfur in 2,6-lutidine to generate phosphate compounds. Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylamine/tetrahydrofuran, room temperature, 1 hr). The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared. The compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 hr and then are further deprotected by heating at about 50° C. overnight to yield preferred antisense compounds.

The pharmaceutical preparations are made following conventional techniques of a pharmaceutical chemist involving mixing, granulating and compressing, when necessary, for tablet forms or mixing, filling, and dissolving the ingredients, as appropriate, to give the desired oral or parenteral products.

Doses of the present compounds (in a pharmaceutical dosage unit as described above) will be an efficacious, nontoxic quantity selected from the range of 1 ng/kg to 500 mg/kg of active compound, preferably less than 10 mg/kg. The selected dose is administered to a human patient in need of inhibition of β₁-AR-specific mRNA expression from 1-6 or more times daily, orally, rectally, by injection, or continuously by infusion. Oral formulations would generally require somewhat larger dosages to overcome the effects of gastric decomposition. Intravenous or intra-arterial administration would generally require lower doses since the drug is placed directly into the systemic circulation. Dosages for nasal sprays typically range from about 10 mg to about 50 mg (total) or about 0.1 mg/kg to about 10 mg/kg. Therefore, the dose will depend on the actual route of administration.

Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence (i.e. in these illustrative examples the rat and human sequences) and determination of secondary structure, T_(m), binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. The results of these analyses are shown in Table 1, for illustrative oligonucleotides and selected target sequences identified in a variety of mammalian and non-mammalian sources.

Highly preferred target regions of the mRNA, are those that are at or near the AUG translation initiation codon, and those sequences that were substantially complementary to 5′ regions of the mRNA. These secondary structure analyses and target site selection considerations were performed using v.4 of the OLIGO primer analysis software (Rychlik, 1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

The sequences of the oligonucleotides given above are highly preferred sequences for inhibiting mammalian β₁-AR polypeptide activity in a host cell. Additional nucleotides at either end derived by the same process (using OLIGO and BLAST) are also envisioned by this invention. The effective lengths of the preferred oligonucleotides are preferably from at least 9 to about 35 or so nucleotides. The inventors contemplate all oligonucleotide compositions in this range (i.e., those of 9, 10, 11, 12, 13, 14, 15, 1,6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or so bases in length) are highly preferred for the practice of the oligonucleotide-based methods of the invention. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense ODN, in which the phosphate substituents are replaced by phosphorothioates. Likewise, one or both ends of the oligonucleotide may be substituted by one or more acridine derivatives that intercalate between adjacent basepairs within a strand of nucleic acid.

4.2 Methods for Screening Patients at Risk for Hypertension

Because mammalian β₁-AR polypeptide plays a key role in hypertension in an animal, it is often desirable to measure and quantitate levels of β₁-AR polypeptide in an animal under a variety of conditions, even during the course of a treatment regimen designed to ameliorate the hypertensive condition in such an animal. Likewise, in many instances, it is desirable to employ methods for screening polymorphisms of the gene encoding mammalian β₁-AR, to identify patients “at risk” for hypertension, and to identify alleles of the gene both in vitro and in vivo.

As such, the use of one or more of the nucleotide compositions described herein as a probe for identifying a gene encoding mammalian β₁-AR, and methods for correlating the presence of such nucleotide segments with the risk of hypertension is particularly desirable.

4.3 Co-Administration of Small Molecule and Peptide Inhibitors

As described herein, in certain embodiments it may be desirable to co-administer one or more of the antisense compositions with one or more pharmaceuticals. For example, one or more of the commercially available anti-hypertensive agents may be co-administered in a particular therapeutic regimen. Such pharmaceuticals include, but are not limited to, amiloride, amlodipine, benazepril, bepridil, candesartan, captopril, chlorothiazide, chlorthalidone, dichlorphenamide, diltiazem, enalapril, ethacrynicacid, felodipine, fosinopril, furosemide, hydrochlorothiazide, hydroflumethiazide, irbesartan, isradipine, lisinopril, losartan, methylclothiazide, metolazone, misoldipine, moexipril, nicardipine, nifedepine, nimodipine, polythiazide, quinapril, ramipril, spironolactone, torsemide, trandolapril, triamterene, valsartan, and verapamil.

Additional small molecular weight ACE inhibitory compounds and oligopeptides, such as those described in U.S. Pat. No. 5,552,397; U.S. Pat. No. 5,449,661; U.S. Pat. No. 5,348,978; U.S. Pat. No. 5,238,921; U.S. Pat. No. 5,098,887 and U.S. Pat. No. 4,216,209 (each specifically incorporated herein by reference in its entirety). In certain embodiments, such small molecules or other anti-hypertensive agents may also be co-administered to an animal along with one or more of the disclosed antisense constructs.

The administration of such anti-hypertensive agents or small molecular weight inhibitors is well known to those of skill in the art, and particular, to health practitioners who routinely diagnose and/or treat animals or patients suffering from hypertension.

4.4 Pharmaceutical Compositions

Therefore, in certain embodiments, the present invention also concerns formulation of one or more of the antisense polynucleotide compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of antihypertensive therapy.

It will also be understood that, if desired, the nucleic acid segment, RNA, DNA or PNA antisense compositions disclosed herein may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents. As long as the composition comprises at least one β₁-AR-specific mRNA inhibitory antisense oligonucleotide, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The RNA, DNA, or PNA-derived antisense compositions may thus be delivered along with various other agents as required in the particular instance. Such RNA, DNA, or PNA antisense compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may comprise substituted or derivatized RNA, DNA, or PNA compositions. Such compositions may include modified peptide or nucleic acid substituent derivatives, as long as the base sequence of the RNA, DNA, or PNA molecule corresponds to one or more of the contiguous base sequences described herein that specifically bind to β₁-AR-specific mRNA, and that reduce or inhibit the extent of translation of this mRNA into biologically-active β₁-AR polypeptides.

The formulation of pharmaceutically-acceptable excipients and carrier solutions are well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.

4.4.1 Injectable Delivery

Alternatively, the pharmaceutical compositions disclosed herein may be administered parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U.S. Pat. No. 5,543,158, U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free-base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying. agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

4.4.2 Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the antisense compositions of the present invention into suitable host cells. In particular, the antisense oligonucleotide compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-lives (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516, specifically incorporated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety).

Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC12 cells (Renneisen et al., 1990; Muller et al., 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 1979) into a variety of cultured cell lines and animals. In addition, several successful clinical trails examining the effectiveness of liposome-mediated drug delivery have been completed (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al., 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature, and results in an increase in permeability to ions, sugars, and drugs.

In addition to temperature, exposure to proteins can alter the permeability of liposomes. Certain soluble proteins such as cytochrome c bind deform and penetrate the bilayer, thereby causing changes in permeability. Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes. For example, MLVs are moderately efficient at trapping solutes, but SUVs are extremely inefficient. SUVs offer the advantage of homogeneity and reproducibility in size distribution, however, and a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs.

Alternatively, the invention provides for pharmaceutically acceptable nanocapsule formulations of the polynucleotide compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated herein by reference in its entirety). In particular, methods of antisense oligonucleotide delivery to a target cell using either nanoparticles or nanospheres (Schwab et al., 1994; Truong-Le et al., 1998) are also particularly contemplated to be useful in formulating the disclosed compositions for administration to an animal, and to a human in particular.

4.5 Therapeutic and Diagnostic Kits

The invention also encompasses one or more of the antisense compounds together with one or more pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and/or other components, such as additional antihypertensive agents, oligonucleotides, peptides, antigens, or other therapeutic compounds as may be employed in the formulation of particular oligonucleotide or polynucleotide delivery formulations, and in the preparation of antihypertensive agents or cardiac therapy for administration to an animal.

As such, preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans. Other preferred animals include primates, sheep, goats, bovines, equines, porcines, lupines, canines, and felines, as well as any other mammalian species commonly considered pets, livestock, or commercially relevant species. The composition may include partially or significantly purified antisense compositions, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing nucleic acid segments encoding such additional active ingredients.

Therapeutic kits may also be prepared that comprise at least one of the β₁-AR mRNA-specific antisense oligonucleotides and polynucleotides disclosed herein and instructions for using the composition as a therapeutic agent. The container means for such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which the antisense composition(s) may be placed, and preferably suitably aliquoted. Where a second antihypertensive agent is also provided, the kit may also contain a second distinct container means into which this second composition may be placed. Alternatively, the plurality of antihypertensive compositions may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container means. The kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained.

Alternatively, for the preparation of diagnostic kits, and for methods relating to the use of these compounds in the identification of β₁-AR polypeptide-encoding nucleic acids in a biological sample, such kits may be prepared that comprise at least one of the β₁-adrenoceptor mRNA-specific antisense oligonucleotides disclosed herein and instructions for using the composition as a probe for β₁-AR -specific nucleic acids in a hybridization assay. The container means for such kits may typically comprise at least one vial, test tube, microcentrifuge tube, or other container means, into which the antisense composition(s) may be placed and suitably aliquoted. Where a radiolabel or fluorigenic label or other such detecting means is included within the kit, the labeling agent may be provided either in the same container as the oligonucleotide composition, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the oligonucleotide composition and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.

4.6 Peptide Nucleic Acid Compositions

In certain embodiments, the inventors contemplate the use of peptide nucleic acids (PNAs) in the practice of the methods of the invention. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, 1997). PNAs may be utilized in a number of methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. An excellent review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (1997) and is incorporated herein by reference. As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of the β₁-adrenoceptor-specific mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of β₁-adrenoceptor-specific mRNA, and thereby alter the level of β₁-adrenoceptor polypeptide in a host cell to which such PNA compositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., 1993; Hanvey et al., 1992; Hyrup and Nielsen, 1996; Nielsen, 1995). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1992) or Fmoc (Bonham et al., 1995) protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used (Christensen et al., 1995).

PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass., USA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., 1995).

4.7 Methods of Nucleic Acid Delivery and DNA Transfection

In certain embodiments, it is contemplated that one or more RNA, DNA, PNAs and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell. Technology for introduction of PNAs, RNAs, and DNAs into cells is well known to those of skill in the art.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Wong and Neumann, 1982; Fromm et al., 1985; Tur-Kaspa et al., 1986; Potter et al., 1984; Suzuki et al., 1998; Vanbever et al., 1998), direct microinjection (Capecchi, 1980; Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990; Klein et al., 1992), and receptor-mediated transfection (Curiel et al., 1991; Wagner et al., 1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Moreover, the use of viral vectors (Lu et al., 1993; Eglitis and Anderson, 1988; Eglitis et al., 1988), including retroviruses, baculoviruses, adenoviruses, adenoassociated viruses, vaccinia viruses, Herpes viruses, and the like are well-known in the art, and are described in detail herein.

4.8 Expression Vectors

The present invention contemplates an expression vector comprising at least one β₁-AR -specific polynucleotide of the present invention. Thus, in one embodiment an expression vector is constructed with a specific DNA molecule orientated in the antisense direction. In another embodiment, a promoter is operatively linked to a sequence region that encodes a functional RNA such as a tRNA, a ribozyme or an antisense RNA.

As used herein, the term “operatively linked” means that a promoter is connected to a functional RNA in such a way that the transcription of that functional RNA is controlled and regulated by that promoter. Means for operatively linking a promoter to a functional RNA are well known in the art.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depend directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the functional RNA to which it is operatively linked.

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

4.9 In Vivo Delivery and Treatment Protocols

To introduce the antisense constructs to cells in vivo, one of any number of conventional ways may be employed. These methods include viral-mediated delivery using retroviral, adenoviral, or adeno-associated viral vectors, and are well known to those of skill in the antisense therapeutic arts.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the U.S. human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka and McLaughlin, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs (FIGS. 2A, 2B and 2C). There are two major genes in the AAV genome: rep and cap. The rep gene encodes proteins responsible for viral replications, whereas the cap gene encodes the capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three viral promoters have been identified and named p5, p19, and p40, according to their map position. Transcription from p5 and p19 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat and Muzyczka, 1984).

Other viral vectors may also be employed as expression constructs in the present invention for the delivery of β₁-AR mRNA-complementary oligonucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polioviruses and herpesviruses may be employed. They offer s several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990).

In order to effect expression of the β₁-adrenoceptor mRNA-complementary antisense sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states, and in particular, in the treatment of hypertension and β₁-AR-related disorders. As described above, the preferred mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.

Once the expression construct has been delivered into the cell the nucleic acid encoding the β₁-adrenoceptor mRNA-complementary oligonucleotide antisense sequences may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the antisense construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

In one embodiment of the invention, the expression construct comprising one or more β₁-AR mRNA-complementary oligonucleotide or polynucleotide antisense sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Reshef (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e. ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.

In a further embodiment of the invention, the expression construct comprising an β₁-AR mRNA-complementary oligonucleotide antisense sequence may be entrapped in one or more nanocapsules, liposomes, or other lipid based DNA delivery agent. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures, and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs that may be employed to deliver an antisense polynucleotide into a target cell include receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1993). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Eur. Pat. Appl. Publ. No. EP0273085, specifically incorporated herein by reference in its entirety).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues. Anderson et al., U.S. Pat. No. 5,399,346, and incorporated herein in its entirety, disclose ex vivo therapeutic methods.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light, of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 Example 1 Antisense Inhibition of β₁-AR mRNA

β₁-antisense, through specific inhibitions of β₁-AR expression, decreases the functional sensitivity of β₁-AR-mediated responses in the face of sympathetic activation and thereby achieves an antihypertensive effect. In this example, an AS-ODN was designed complementary to rat β₁-AR mRNA and its ability was demonstrated to inhibit β₁-AR density and function in the heart and to reduce BP in spontaneously hypertensive rats.

5.1.1 Methods

5.1.1.1 Antisense Design and Administration

AS-ODN and inverted ODN control were 15-mer and targeted to the AUG start codon of rat β₁-AR mRNA (Machida et al., 1990). The sequence of AS-ODN is 5′-CCGCGCCCATGCCGA-3′ (SEQ ID NO:195), and the inverted ODN is 5′-AGCCGTACCCGCGCC-3′ (SEQ ID NO:196). This AS-ODN was chosen from 6 AS candidates targeted to different regions of β₁-AR mRNA on the basis of the intensity of cardiac β₁-AR inhibition and reduction of BP in SHRs. These oligonucleotides were modified by backbone phosphorothioation. ODNs delivered with cationic liposomes were injected into the tongue vein.

5.1.1.2 Preparation of Liposomes and ODN/Liposome Complex

The cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) was mixed with helper lipid 1-α-dioleoyl phosphatidylethanolamine (DOPE, Avanti Polar Lipids, Alabaster, Ala.) at a 1:1 molar ratio, briefly sonicated, and stored at 4° C. until use. The average diameter of liposomes is 200 to 300 nm (Tang and Hughes, 1998). ODN/liposome complex was prepared on the day of use by mixing the desired amounts of ODNs with DOTAP/DOPE to the final DNA concentration of 300 μg/mL in 5% wt./vol. dextrose in water and incubating at room temperature for 60 min. Two DNA/lipid molar ratios, i.e. 1:0.5 and 1:2.5, were used in the studies.

5.1.1.3 Animal Surgery

Adult male SHRs (250 to 350 g, Harlan, Indianapolis, Ind.) were kept in cages in a room with a 12-hr. light-dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum.

5.1.1.4 Telemetric Sensor Implantation

Before implantation, the zero of each radiotransmitter (TA11PA-C40, Data Sciences, St. Paul, Minn.) was verified to be ≦4 mm Hg. SHRs were anesthetized with 100 mg/kg ketamine and 15 mg/kg xylazine, and a midline abdominal incision was made. A fluid-filled sensor catheter was then inserted into the right femoral artery, and the tip of the catheter was in the abdominal aorta caudal to the renal arteries. The rats with implants were allowed to recover for 1 week.

5.1.1.5 Jugular Vein Cannulation

One week after telemetric implantation, rats were anesthetized, and a curved catheter made of PE 50 and vinyl tubing was inserted into a curved catheter made of PE 50 and vinyl tubing was inserted into the jugular vein. The tubing was led under the skin of the neck and exposed on the back to allow for drug infusion. Rats were allowed to recovery for 24 hr before experimentation. The catheters were flushed with 100 U heparin every day to prevent clogging.

5.1.1.6 Membrane Preparation and β-AR Binding Assay

Four days after intravenous injection of saline (n=6) or 1 mg/kg inverted ODN (n=6) or 2, 4, 10 or 18 days after injection of 1 mg/kg β₁-AS-ODN (n=24), animals were euthanized, and membranes were prepared from heart ventricles as previously described (Baker and Pitha, 1982). For saturation experiments, 100 μg membrane protein was incubated in triplicate with 6 concentrations of [¹²⁵I](⁻)-iodocyanopindolol (ICYP, NEN Life Science, 6.25 to 100 pmol/L) in a total volume of 250 μL containing 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L MgCl₂ at 36° C. for 60 minutes. The nonspecific and β₂-AR-binding levels were determined in the presence of 1 μmol/L (±)-alprenolol and 150 nmol/L CGP20712A (RBI), respectively. Then the reaction mixture was passed through Whatman (GF/B glass fiber filter using a Brandel harvester, and the bound radioactivity was counted for 1 min.

5.1.1.7 Tissue Preparation and Quantitative Autoradiography

Four days after injection of 1 mg/kg β₁-AS-ODN (n=6) or saline (n=6), rats were killed, and tissues were removed and frozen in dry ice. Coronal sections of brain, horizontal sections of heart, and sagittal sections of kidney (20 μm) were cut on a cryostat (Microm, Thornwood, N.Y.) at ⁻20° C. and mounted on microscope slides. Every seventh slide was stained with hematoxylin and eosin for histology. Tissue sections were preincubated in Krebs buffer (mmol/L: NaCl 118.4, KCl 4.7, MgSO₄ 1.2, CaCl₂ 1.27 and NaH₂PO₄ 10.0, pH 7.1) containing 0.1 mmol/L GTP, 0.1 mmol/L ascorbic acid, and 10 μmol/L PMSF for 30 minutes at 25° C. Sections were then incubated in Krebs buffer containing 0.1 mmol/L ascorbic acid and 10 μmol/L PMSF with 100 pmol/L ICYP at 25° C. for 150 minutes in the presence of 1 μmol/L (⁻) propranolol, 100 nmol/L ICI 118,551 (β₁-selective antagonist), or 100 nmol/L CGP20712A (β₁-selective antagonist) to distinguish nonspecific, β¹⁻, and β₂-bindings. Labeled sections were rinsed in the same buffer, followed by two 15-min. washes at 37° C. in the buffer, and rinsed in distilled water at 25° C. (Matthews et al., 1994). Dried sections were then exposed to x-ray films. The images were quantified with a computerized image analysis system (MCID, Imaging Research) and normalized with ¹²⁵I standards. Nonspecific binding was <10% of total binding.

5.1.1.8 Determination of Effects of β₁-AS and Atenolol on Cardiovascular Parameters in Response to β-Stimulation

48 hr after injection of 1 mg/kg β₁-AS-ODN (n=9) or inverted ODN (n=6), SHRs were anesthetized and killed. Hearts were quickly removed and perfused via the aorta with oxygenated Krebs buffer (118 mmol/L NaCl, 18.75 mmol/L NaHCO₃ 1.2 mmol/L KH₂PO₄, 4.7 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.25 mmol/L CaCl₂, 11.1 mmol/L glucose, and 0.01 mmol/L EDTA) at a constant flow of 7.0 mL/min. at 36° C. Coronary perfusion pressure was measured via a catheter placed proximal to the aorta and connected to a pressure transducer (Gould Staham P23ID, Eastlake, Ohio). A latex balloon filled with water and connected to the pressure transducer was inserted into the left ventricle through the left atrium to measure left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), and developed left ventricular pressure (dLVP) (dLVP=LVSP−LVEDP). LVEDP during equilibration was set at 5 to 7 mm Hg. Coronary perfusion pressure, LVEDP, and LVSP were recorded continuously on a 4-channel recorder (Astro-Med, West Warwick, R.I.). After baseline values for dLVP and heart rate (HR) were stable for 5 min., isoproterenol (ISO, nonspecific β-agonist) was given at 0.01, 0.025, 0.05, and 0.12 μmol/L at 10 min. intervals so as to avoid the effect of tachyphylaxis.

The effects of β₁-AS-ODN and atenolol on cardiac dP/dt_(max) and systolic blood pressure (SBP) were compared in the same group of SHRs (n=4). Two days after catherization of the jugular vein, control values were taken and 1 mg/kg β₁-AS-ODN was injected. 48 hr later, rats were tested for the effect of β₁-AS-ODN. The rats were allowed to recover until all the cardiovascular parameters returned to control values. Then 1 mg/kg atenolol (β₁-selective antagonist) was injected, and rats were tested 30 min. later. For β₁-stimulation, SHRs were infused with dobutamine (β₁-selective agonist) through a jugular vein catheter at 5, 10, 20, and 40 μg·kg⁻¹·min.⁻¹. Each dose was given for 5 min. continuously and at 1-hr intervals until all the cardiovascular parameters returned to baseline values so as to avoid the effect of tachyphylaxis. BP and HR were sampled every 1 min. dP/dt_(max) was calculated from the slope of the rising pulse-pressure curve and determined every 1 min. The difference between values at each dose and baseline was denoted as Δ.

5.1.1.9 BP Monitoring

Each rat cage was placed on a receiver (RLA 1020, Data Sciences, St. Paul, Minn.) for measurement of cardiovascular parameters. Data were collected with a computer-based data acquisition program (Dataquest Lab-PRO3.0; Data Sciences). BP and HR were measured every 10 min. and averaged every 1 to 24 hrs. Before treatment, SHRs were monitored for a week to get a stable baseline.

Rats were warmed for 20 to min. in cages on heating pads. The temperature was controlled at 35° C. to 37° C. Then the rats were placed in a plastic restrainer kept at 37° C. A pneumatic pulse sensor was attached to the tail. After cuff inflation, SBP was determined as the first pulsatile oscillation on the descending side of the pressure curve. HR was determined by manual counting of pulse numbers per unit time. BP and HR were recorded by a Narco physiograph. Data values of each rat were taken as an average of at least 4 stable readings. Baseline was determined by averaging 3 days of measurements before antisense administration.

5.1.1.10 Statistical Analysis

Values were expressed as mean±SEM. The difference was considered statistically significant at P<0.05. An unpaired t test was used to compare B_(max), dLVP, and BP in 2 groups. One-way repeated-measures ANOVA and Tukey test were used to compare ΔdP/dt and ΔHR on dobutamine infusion in different groups. Pearson product-moment correlation was used to assess the relationship between β₁-AR B_(max) and dLVP.

5.1.2 Results

5.1.2.1 Effect on Cardiac β-AR Density

FIG. 1A shows that a single intravenous injection of 1 mg/kg β₁-AS-ODN delivered with cationic liposomes at a 1:2.5 molar ratio significantly reduced the β₁-AR density in the SHR hearts for 18 days (P<0.01). The drop was at its maximum of 470/0 on day 4, 33% on day 10 and maintained at 29% on day 18. In contrast, there was no significant change in the B_(max) of β₂-ARs. The K_(D) of both subtypes remained unaltered (Table 1). Consequently, the β₁/β₂ subtype ratio in the ventricles was diminished from ≈70/30 to ≈50/50 by β₁-AS-ODN. Inverted ODN had no effect on either subtype (FIG. 1B).

5.1.2.2 Effect on Cardiac Contractility and HR in Response to β-Stimulation

48 hr after injection of 1 mg/kg β₁-AS-ODN, the cardiac inotropic and chronotropic responses to β-stimulation were determined in SHRs in vitro and in vivo.

First, isolated hearts were perfused with incrementing doses of ISO, which enhanced HR and contractility via activating β-ARs. The dLVP-ISO dose-response curve, which reflected the positive inotropic effect of ISO, was significantly shifted downward by β₁-AS-ODN (P<0.02). HR was not significantly decreased, except at 1 point, i.e. 0.01 μmol/L ISO (P<0.05) (FIG. 2A and FIG. 2B).

An in vivo test was performed in conscious SHRs monitored by radiotelemetry. β-AS-ODN significantly (P<0.02) dampened the increase in dP/dt_(max) in the face of dobutamine (β₁-selecrive agonist) (FIG. 3A). The change in HR was not significantly reduced (FIG. 3B), which echoed the results in isolated perfused hearts. Conversely, the response of BP to dobutamine was biphasic (FIG. 3C). Dobutamine elevated SBP by 5 to 8 mm Hg at low infusion speed and reduced SBP at higher speed, probably as a result of the partial β₂-agonistic activity of dobutamine at high doses and the consequent vasodilatory effect on BP. FIG. 3 also compares the results with β₁-AS-ODN and atenolol (β₁-selective antagonist). Relative to β₁-AS-ODN, 1 mg/kg atenolol produced a more profound decline in ΔdP/dt_(max) and ΔHR during a 5 hr. period of time after injection. Moreover, it reduced basal dP/dt_(max) (2450±295 mm Hg/s versus control, 2937±277 mm Hg/s) and caused bradycardia (295±12 bpm versus control, 365±8 bpm) (P<0.05), whereas β₁-AS did not change basal contractility (2922±249 mm Hg/s) or HR (365±12 bpm). But the effects of atenolol on ΔdP/dt_(max) and ΔHR were transient. Within 24 hr after atenolol administration, the inotropic and chronotropic effects of dobutamine had returned to control levels.

TABLE 3 B_(MAX) AND K_(D) OF β₁- AND β₂-ARs IN CARDIAC VENTRICLES OF SHRs 4 DAYS AFTER TREATMENT WITH SALINE, INVERTED ODN, OR β₁-AS-ODN β₂ Total (β₁+β₂) β₁ B_(max), K_(D), pmol/L B_(max), fmol/mg K_(D), pmol/L B_(max), fmol/mg K_(D), pmol/L fmol/mg Saline 55.4 ± 6.7 27.6 ± 0.9 64.7 ± 5.4 18.7 ± 0.9 29.6 ± 0.5 9.1 ± 0.6 Inverted 57.1 ± 3.5 26.3 ± 3.0 66.0 ± 1.0 18.1 ± 2.3 32.0 ± 2.4 8.7 ± 0.5 ODN β₁-AS- 37.0 ± 1.0* 19.8 ± 1.8* 60.5 ± 3.4 10.0 ± 1.2* 30.7 ± 3.3 9.9 ± 0.5 ODN Data represent mean ± SEM of each group (n = 6 to 10) *p < 0.01 vs. saline control

5.1.2.3 Effect on BP of SHRs

FIG. 4 shows the effects of a single injection of 1 mg/kg β₁-AS-ODN on the BP of SHRs measured by the tail-cuff method. AS-ODN was delivered with cationic liposomes at different molar ratios of DNA/lipid, i.e. 1:0.5 and 1:2.5. β₁-AS-ODN delivered with liposomes at a 1:0.5 ratio diminished SBP for 8 days. The maximum drop was 38±5 mm Hg. When the molar ratio was increased to 1:2.5, this hypotensive effect was drastically prolonged to 20 days. No effect was seen with inverted ODN.

To compare the effects of β₁-AS-ODN and atenolol, radiotelemetry was used to monitor BP and HR on a regular basis. β₁-AS-ODN 1 mg/kg delivered with liposomes at a 1:0.5 ratio produced a maximum drop of 15 mm Hg in mean BP (FIG. 5A). The antihypertensive effect lasted for 8 days, which was consistent with the results measured with the tail cuff. HR was not significantly altered (FIG. 5B). In contrast to AS-ODN, although the onset of the hypotensive effect caused by 1 mg/kg atenolol occurred as early as 20 min. after injection, it lasted for only 10 hr (FIG. 6A). In addition, atenolol caused considerable bradycardia up to an average of ⁻75 bpm (FIG. 6B).

5.1.2.4 Effect on β-AR Distribution in Brain, Heart, and Kidney

Quantitative autoradiography of 6 to 18 tissue slices in brain, heart, and kidney was analyzed 4 days after intravenous β₁-AS-ODN administration. No changes in the distribution of β-ARs in the forebrain and brain stem regions were detected. This indicated the absence of antisense effect on the β-AR expression in CNS. However, β₁-AS-ODN significantly (P<0.05) reduced β₁-AR density in cardiac ventricles (from 30.2±2.1 to 20.6±2.5 fmol/mg) and renal cortex (from 26.4±3.1 to 17.4±3.3 fmol/mg). This was consistent with the binding results. β₂-ARs were not affected in any tissues.

5.1.3 Discussion

This example compares the effects of a novel β₁-AS-ODN on high BP in a model of hypertension with a currently used β-blocker. β₁-AS-ODN knocked down β₁-adrenergic activity, resulting in long-term attenuation of BP. The results indicated that β₁-AS-ODN reduced β₁-AR density and cardiac contractility after β₁-stimulation in vitro and in vivo and lowered high BP of SHRs. These findings are consistent with the hypothesis that β₁-AS, through the inhibition of β₁-AR expression, is able to render heart, kidney, and other tissues less sensitive to sympathetic activation, which is a major contributing factor in high BP. A single injection of β₁-AS-ODN effectively decreased the cardiac β₁-AR density, which was accompanied by diminished ventricular contractility and cardiac output in response to β-stimulation. The antihypertensive effect in SHRs was up to a 38 mm Hg reduction lasting as long as 20 days.

Treatment of β₁-AS-ODN reduced cardiac β₁-AR density by ≈50% in 4 days. Considerable variation is reported in the literature on the half-life of β₁-ARs (Baker and Pitha, 1982; Neve and Molinoff, 1986 and Winter et al., 1988). From the results with the AS-ODN inhibition, the half-life of cardiac β₁-ARs is ≈2 to 4 days to allow for 50% reduction of β-AR density within 4 days.

Both tail-cuff and telemetry measures of BP showed a significant drop after antisense treatment. SHRs responded to β₁-AS-ODN to a greater degree of hypotension when subjected to tail cuff vs. telemetry. In addition, the baseline measured with the tail cuff was consistently higher than that with telemetry by 20 to 30 mm Hg in the same rats. Bazil et al., 1993 reported a similar phenomenon. They compared the cardiovascular parameters recorded by telemetry, tail cuff, and arterial catheter and observed a more sensitive hypotensive effect of captopril with tail cuff. Tail-cuff measurement of BP involves warming and restraint of rats. It is conceivable that β₁-AS, through the suppression of sympathetic activity, can decrease the BP of animals under stress more effectively.

Cationic liposomes are effective vehicles for gene delivery. It has been shown that liposomes entrapment not only improves the cellular uptake of DNA but also protects DNA from degradation and extends its circulation time (Allen, 1997; Liu et al., 1997). Numerous factors influence the efficiency of cationic liposome-mediated intravenous gene delivery, such as DNA/lipid ratio, selection of lipids, and preparation procedure. A widely used lipid formula, DOTAP/DOPE, was used to deliver β₁-AS-ODN at 4 molar ratios. The optimal ratio of 1:2.5 was determined on the basis of the duration and magnitude of the antihypertensive effects. A profound and prolonged fall in BP of 38 mm Hg up to 20 days was achieved at this ratio, which followed the reduction in β₁-AR binding to a maximum of 47% at day 4, 33% at day 10, and 29% at day 18. This implies that β₁-AS-ODN effectively inhibits the functionally active receptors involved in the BP.

The blood-brain barrier formed by capillary endothelia is permeable only to small lipophilic molecules with a molecular weight of <600 Da (reviewed by Pardridge, 1998). Owing to their high hydrophilicity, ODNs undergo negligible transport through blood-brain barrier and have very limited access to CNS. The cellular and organ distributions of DNA/liposome complexes with fluorescent labeling were previously studied in mice after intravenous injection, and the results indicated that the complexes were taken up primarily by capillary endothelial cells in most of the peripheral organs, including lung, heart, kidney, and spleen, but were absent in the brain (McLean et al., 1997). Although brain retention of liposomes after peripheral administration was observed in some cases, it was due to entrapment within the brain microvasculature (Schackert et al., 1989). Autoradiography in brain revealed no detectable changes in the expression and distribution of β-ARs after intravenous β₁-AS-ODN injection. This provided further evidence that the use of antisense did not have CNS effects.

High specificity based on gene sequence has made antisense an increasingly useful tool in numerous studies and clinical trials. Its success is manifested by the recent approval of the first antisense drug, Vitavene, by the Food and Drug Administration. However, sequence-independent interactions have also been reported with AS-ODNs. High doses are usually responsible for the nonspecific effects (Chang et al., 1989), but another possible reason is that currently available databases do not cover every gene; thus, homology comparison by BLAST search may no guarantee sequence specificity of AS-ODNs. In this study, β₁-AS-ODN inhibited β₁-AR expression without changing β₂-ARs. Although this is likely due to the specificity of the β₁-AS-ODN sequence, other possibilities remain, such as indirect effects on regulatory mechanisms.

In patients with chronic heart failure, the severity of the disease closely relates to the decrease in cardiac β-AR density and functional responsiveness (Brown et al., 1992). In SHRs treated with β₁-AS-ODN, a marked attenuation of the β₁-AR-mediated positive inotropic response in vitro and in vivo, concurrent with the diminished cardiac β₁-AR level. Therefore, these results indicated a positive correlation between cardiac β₁-AR number and functional sensitivity (correlation coefficient>0.90, P<0.01).

Despite the large decrease in BP, no reflex tachycardia was observed after antisense treatment. However, the suppressive effect of β₁-AS-ODN on Hr was less significant than its negative inotropic response. Several possibilities can be considered. First, β₁-AS-ODN did not affect β₂-ARs, which played an important role in the regulation of HR (Kaumann, 1986; Rodefeld et al., 1996), although it was not involved in the cardiac contraction. Second, antisense inhibition of β₁-AR expression is gradual and less extensive than with β-blockers. It is also possible that β₁-ARs may have a larger reserve for controlling HR than contractility.

5.2 Example 2 Prolonged Reduction in High Blood Pressure With β₁ AR Oligodeoxynucleotides

Since the introduction of propranolol in 1965, β-blockers have become major first-line drugs for hypertension. Through the inhibition of β-adrenergic receptors in heart and kidney, β-blockers lower high blood pressure via the reduced response to the sympathetic nervous system. However, all current β-blockers have to be taken daily. Also, most have central nervous system side effects that lead to poor patient compliance. Furthermore, the mechanism of β-blockade in hypertension is not well understood (Man in't Veld et al., 1988). Antisense oligonucleotides have been successfully constructed to components of the renin-angiotensin system (RAS) to decrease blood pressure (Phillips et al., 1994). In view of this, novel antisense oligonucleotides targeted to β₁-adrenergic receptors (β₂-ARs), or the brain. Therefore, it is likely to have fewer side effects and longer-lasting action.

It has been previously shown that antisense oligodeoxynucleotide (β₁-AS-ODN) significantly inhibits β₁-AR expression in the cardiac ventricles, which results in suppressed inotropic response to adrenergic activation and thereby contributes to hypotension. In addition to inducing positive inotropy and chronotropy in the heart, β₁-ARs are also responsible for mediating the sympathetic stimulation of renin expression and secretion from juxtaglomerular cells of the renal cortex. Infusion of isoproterenol has been shown to increase renin expression and secretion and plasma renin activity (PRA) in rats (Holmer et al., 1997). β-Blockers reduce PRA in patients, (Blumenfeld et al., 1999). However, despite the evidence that β-blockers are more effective in patients with high renin profiles (Buhler, 1988), the importance of β-blocker-induced decreases in renin release has been debated (Man in't Veld et al., 1983). The present study investigates whether β₁-AS-ODN reduces renin expression and secretion and whether the RAS is involved in the antihypertensive impact of β₁-AS-ODN could be improved by delivery with cationic liposomes and to determine the optimal charge ratio of liposome:ODN.

5.2.1 Methods

5.2.1.1 Antisense Sequence and Delivery

AS-ODN and inverted ODN control were 15-mer and targeted to the AUG start codon of rat β₁-AS-ODN is 5′-CCGCGCCCATGCCGA-3′ (SEQ ID NO:197), and the inverted ODN is 5′-AGCCGTACCCGCGCC-3′ (SEQ ID NO:198). These ODNs were modified by backbone phosphorothioation. The cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) mixed with the helper lipid L-α-dioleoyl phosphatidylethanolamine (DOPE, Avanti Polar Lipids) at 1:1 molar ratio was used to deliver ODNs in a single intravenous injection into the tongue vein. ODN-liposome complex was prepared on the day of use by mixing desired amounts of ODNs with DOTAP/DOPE to a final DNA concentration of 300 μg/mL in 5% (wt./vol.) dextrose in water and incubating at room temperature for 60 min.

5.2.1.2 Animals

Adult male SHR (4 to 6 mos. old, Harlan, Indianapolis, Ind.) were kept in cages in a room with a 12 hr. light-dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum. Tail blood was collected for determination of PRA and angiotensin II (Ang II) levels.

5.2.1.3 Blood Pressure Measurement

Blood pressure was measured by the tail-cuff method as described above. Systolic blood pressure (SBP) was determined as the first pulsatile oscillation on the descending side of the pressure curve. Data values of each rat were taken as an average of ≧4 stable readings. Baseline was determined by averaging 3 days of measurements before antisense administration.

5.2.1.4 Membrane Preparation and β-Adrenergic Receptor Binding Assay

Four days after intravenous injection of saline (n=6) or 0.5 mg/kg inverted ODN (n=6) or 4, 10, 18 and 40 days after injection of 0.5 mg/kg β₁-AS-ODN (n=24), animals were euthanized, and membranes were prepared from the renal cortex of the left kidneys as previously described (Baker and Pitha, 1982). For saturation studies, 100 μg membrane protein was incubated in triplicate with 6 concentrations of [¹²⁵](−)iodocyanopindolol (ICYP, NEN Life Science, 6.25 to 100 pmol/L) in a total volume of 250 μL containing 50 mmol/L Tris-HCl (pH 7.4) and 5 mmol/L MgCl₂ at 36° C. for 60 min. The nonspecific and β₂-adrenergic receptor binding levels were determined in the presence of 1 μmol/L (±)-alprenolol and 150 mmol/L CGP20712A (RBI), respectively. Then the reaction mixture was passed through a Whatman GF/B glass fiber filter with a Brandel harvester, and the bound radioactivity was counted for 1 min.

5.2.1.5 Tissue Preparation and Quantitative Autoradiography

Four days after injection of 0.5 mg/kg β₁-AS-ODN (n=6) or saline (n=6), rats were euthanized, and the right kidneys were removed and frozen in dry ice. Sagittal sections of kidney (20 μm) were cut on a cryostat (Microm) at −20° C. and mounted on microscope slides. Every seventh slide was stained with hematoxylin and eosin for histology. Receptor autoradiography was performed as described (Matthews et al., 1994) with 100 pmol/L ICYP at 25° C. for 150 min. in the presence of 1 μmol/L (−)-propranolol, 100 mmol/L ICI 118,551 (β₂-selective antagonist), or 100 nmol/L CGP 20712A (β₁-selective antagonist) to distinguish nonspecific, β₁, and β₂-bindings. The images were quantified with a computerized image analysis system (MCID, Imaging Research) and normalized with ¹²⁵I standards. Nonspecific binding was <10% of total binding.

5.2.1.6 Reverse Transcription-Polymerase Chain Reaction and Southern Blotting

At different time points after the single injection of β₁-AS-ODN or inverted ODN, rats were euthanized, and the renal cortex was dissected from the left kidneys, immediately dipped into RNA later tissue storage buffer (Ambion), and stored at −20° C. Total RNA was extracted with RNAwiz reagent (Ambion) and quantified by spectrophotometer. RNA samples from 4 to 5 rats from each time point were pooled. RNA (5 μg) was digested by DNase I and reverse-transcribed by Superscript reverse transcriptase (GIBCO BRL) at 42° C. for 50 min. and 1/20 of the reverse transcription (RT) product was used to run a polymerase chain reaction (PCR) for 20 cycles. PCR primers for β₁-AR were 5′-CTCCGAAGCTCGGCATGG-3′ (SEQ ID NO:199) (forward) and 5′-GCACGTCTACCGAAGTCCAGA-3′ (SEQ ID NO:200) (reverse) and yielded products of 432 bp, which spanned the AUG start codon. Primers for preprorenin were 5′-AGGCAGTGACCCTCAACATTACCAG-3′ (SEQ ID NO:201) (forward) and 5′-CCAGTATGCACAGGTCATCGTTCCT-3′ (SEQ ID NO:202) (reverse) and yielded products of 362 bp.

Primers for GAPDH were 5′-ATCAAATGGGGTGATGCTGGTGCTG-3′ (SEQ ID NO:203) (forward) and 5′-CAGGTTTCTCCAGGCGGCATGTCAG-3′ (SEQ ID NO:204) (reverse) and yielded products of 505 bp (Jo et al., 1996). RT-PCR products were subjected to Southern blotting, hybridized with psoralen-biotin-labeled cDNA probes, and detected with nonisotopic kits (Ambion). After the membranes had been exposed to x-ray films, the intensity of β₁-AR and preprorenin mRNAs was quantified by densitometry and normalized with GAPDH mRNA levels. The studies were repeated at least twice.

5.2.1.7 PRA and Plasma Ang II Levels

PRA was determined with an angiotensin I (¹²⁵I) radioimmunoassay kit (DuPont). Plasma Ang II levels were measured by radioimmunoassay as previously described (Phillips and Kimura, 1988).

5.2.1.8 Statistical Analysis

Values were expressed as mean±SEM. Differences were considered statistically significant at a value of P<0.05. One-way repeated ANOVA and Tukey's test were used to compare blood pressure before and after AS-ODN treatment. Unpaired t test was used to compare B_(max1) PRA, and plasma Ang II levels in 2 groups.

5.2.2 Results

5.2.2.1 Optimization of β₁-AS-ODN Delivery by Cationic Liposomes

Systemic delivery of AS-ODN was optimized with the commercially available cationic lipid DOTAP mixed with neutral lipid DOPE. Previous studies reported that a charge ratio of DOTAP:DNA of ≈2.0 achieved the best gene delivery in vivo and in vitro (Yang et al., 1997 and Templeton et al., 1997). Therefore, 5 charge ratios of DOTAP:ODN were tested ranging from 0 to 3.5 to deliver 0.5 mg/kg β₁-AS-ODN intravenously. It was noticed that different batches of liposome mixture varied slightly in structure and particle size, which may influence the delivery efficiency. FIG. 7 shows the effect of different liposome:ODN charge ratios on blood pressure of SHR (n=6 for each ratio) in a representative experiment. β₁-AS-ODN alone, i.e. at ratio 0, did not change SBP, whereas ratio 0.5 significantly reduced SBP by up to 33 mm Hg for 7 to 8 days. When the ratio was increased, the duration of the hypotensive impact was drastically prolonged to 20 days at ratio 1.5 and 33 days at ratio 2.5 and 3.5, varying with liposome preparations. But the maximum drop in SBP was greater at ratio 1.5 and 2.5 (≈35 mm Hg) than at ratio 3.5 (≈25 mm Hg) (Table 2). Accordingly, the optimal charge ratio of DOTAP:ODN was determined to be 2.5. In the subsequent experiments, SHR (n=24) injected with 0.5 mg/kg β₁-AS-ODN with liposomes at a charge ratio of 2.0 were analyzed for the time course of changes in SBP, receptor levels, and peripheral RAS.

TABLE 4 RANGES OF AMPLITUDE AND DURATION OF REDUCTION IN BLOOD PRESSURE OF SHR AFTER A SINGLE INTRAVENOUS INJECTION OF 0.5 MG/KG β₁-AS-ODN DELIVERED IN DIFFERENT CHARGE RATIOS OF LIPOSOME/ODN Maximum Reduction Range of Charge Ratio in Blood Pressure, Reduction, Duration, d of Liposome/ODN mm Hg mm Hg (Range 0 2 *** *** 0.5 35-38 24-38 7-8 1.5 28-35 18-35 18-20 2.5 30-34 20-34 20-33 3.5 20-24 15-24 20-33

5.2.2.2 Effects of β₁-AS-ODN on β-Adrenergic Receptors in Renal Cortex

Scatchard analysis of β-AR binding in renal cortex (FIG. 8A, FIG. 8B and FIG. 8C) indicated that β₁-AR was the major subtype in the control rats, composing 70% of total β-AR. After β₁-AS-ODN injection, the B_(max) of β₁-ARs was diminished significantly, by 35% on day 4 (P<0.05), 29% on day 10 (P<0.05), and 23% on day 18, and completely restored on day 40. β₁-AR reduction in kidney coincided with that in heart, and both were accompanied by a significant drop in SBP (P<0.0 1) (FIG. 8A). In contrast, the β₂-AR level was not affected (FIG. 8B), nor was the affinity of either subtype. Inverted ODN had no effect on either subtype (FIG. 8C).

Kidney slices from the same rats were subject to quantitative autoradiography to display the structural distribution of β-ARS. β₁-Subtype composed ≈60% of the β-AR levels, which was localized predominantly in the renal cortex and the outer band of the medulla. β₂-Subtype was more diffusely distributed in the kidney at a lower level. This result was consistent with previous reports (Summers et al., 1985). Four days after β₁-AS-ODN treatment, the overall density of β₁-subtype in kidney was significantly reduced from 23.5±2.1 to 15.4±3.3 fmol/mg (P<0.05). The diminution in renal cortex was particularly conspicuous because of the higher basal level. As expected, the distribution and concentration of β₂-subtype remained unchanged in accord with binding results. This further confirms the specificity of the inhibitory effect of β₁-AS-ODN on β₁-subtype.

In an effort to demonstrate whether β₁-AS-ODN decreases the mRNA level of β₁-AR by inducing RNase H digestion, a pair of primers flanking the AUG start codon where β₁-AS-ODN was targeted was used to run a semiquantatitative RT-PCR™ for 20 cycles, followed by Southern blotting. β₁-AS-ODN did not reduce the level of steady-state β₁-AR mRNA in renal cortex, indicating that the inhibition of β₁-AR expression was not at the transcriptional level.

5.2.2.3 Effect of β₁-AS-ODN on Peripherl RAS

RT-PCR™ revealed that the preprorenin mRNA level in renal cortex was transiently decreased to 62% of control 4 days after β₁-AS-ODN injection. It was completely reversed by day 18 (FIG. 9A). Conversely, PRA and plasma Ang II levels showed different patterns of reduction, which were significantly decreased on day 10 and day 18 (P<0.01) but not on day 4. Thus, PRA and Ang II seemed to have as delayed action relative to the reduction in renin mRNA (FIG. 9B).

5.2.3 Summary

Because β₁-adrenergic receptors are also involved in renin expression and secretion from the kidney, the present study was designed to evaluate the effect of β₁-AS-ODN on peripheral RAS and its contribution to the reduction in blood pressure. In addition, a significant improvement of hypotensive action up to 33 days was achieved by optimizing the delivery of β₁-AS-ODN with cationic liposomes.

β-blockers have been used to treat hypertension for 3 decades. The reasons for their antihypertensive effects remain largely unclear, but the inhibition of renin release is regarded as a primary mechanism. Many β-blockers can reduce PRA in patients and experimental animals (Blumenfeld et al., 1999 and Holmer et al., 1994). They are found to be more effective in patients with higher renin profiles (Buhler, 1988). In this study, it was found that β₁-AS-ODN effectively decreased PRA and Ang II in the long term. But the decrease in PRA and Ang II did not occur until ≈10 days after β₁-AS-ODN injection, in contrast to the rapid drop in cardiac output 2 days after injection. Thus, it appears that the effects of β₁-AS-ODN on the kidney renin and the circulating RAS are more delayed than cardiac action. Suppression of cardiac output may account for the early phase of the antihypertensive effect of β₁-AS-ODN, whereas the inhibition of renin-angiotensin activity acts as the secondary mechanism underlying the sustained reduction of blood pressure in SHR.

Receptor binding assay showed that β₁-AS-ODN reduced the β₁-AR levels in renal cortex by ≈30% for 18 days. This is consistent with the decrease of β₁-AR in heart ventricles in magnitude and time course. This suggests that β₁-AS-ODN delivered by cationic liposomes is rapidly transported to peripheral organs after intravenous injection and effectively taken up into heart and kidney cells to a comparable extent. Several mechanisms have been proposed for the AS-ODN inhibition of the expression of target proteins. One involves the decrease in mRNA levels resulting from RNase H digestion of the RNA strand of the RNA-DNA duplex (Phillips et al., 1996 and Phillips et al., 1997). To test this hypothesis, a pair of primers that flanked the AUG start codon of β₁-AR mRNA were designed where AS-ODN bound to perform semiquantitative RT-PCR™. As shown in the Results, there was no reduction in the RT-PCR™ products, indicating the absence of RNase H action. Therefore, the inhibition of β₁-AR expression probably occurs in post-transcriptional steps.

By decreasing β₁-AR levels in kidney, β₁-AS-ODN significantly reduced PRA and the subsequent plasma Ang II levels. This is unlikely to be through the inhibition of renin expression, however, because there is no long-term diminution of renin mRNA levels. Instead, β₁-AS-ODN may exert its inhibitory impact on renin secretion or the conversion of inactive renin to active renin. This hypothesis is consistent with the observation that β-adrenergic stimulation of renin expression had a time course different from that of renin secretion (Holmer et al., 1997 and Chen et al., 1993). Furthermore, β-blockers have been shown to reduce prorenin processing to active renin without changing total renin (prorenin+PRA) levels in plasma (Blumenfeld et al., 1999).

Efficient gene delivery is vital to the therapeutic application of AS-ODN in vivo. Among nonviral vectors, cationic liposomes are the most widely used. They are safe, nonimmunogenic, and east to produce on a large scale. However relatively low transfection efficiency has been obtained after intravenous administration, mainly because of the inactivation of cationic liposome by serum. It was recently shown that increasing the charge ratio (±) of liposome to DNA and inducing the maturation of liposome-DNA complex by prolonging incubation time can overcome this problem (Yang et al., 1997; 1998). The optimal charge ratio of DOTAP:DNA was demonstrated to be ≈2 (Yang et al., 1997 and Templeton et al., 1997). Thus, 5 charge ratios were tested ranging from 0 to 3.5 to optimize the AS-ODN delivery. As shown in the results, increasing the charge ratio not only improved the delivery efficiency but also prolonged the duration of β₁-AS-ODN action. The best antihypertensive result (−35 mm Hg for 33 days) was consistently achieved at ratio 2.5.

In summary, β₁-AS-ODN delivered with cationic liposomes at a single intravenous injection achieves a marked and sustained hypotensive effect (30 to 35 mm Hg for 33 days) in SHR. The β₁-AS-ODN is clearly longer lasting than any current drug, does not inhibit β₂-adrenergic receptors or cross the blood-brain barrier, and has negligible effect on heart rate. Therefore, the antisense is likely to have fewer side effects than currently used β-blockers. Inhibition of cardiac contractility initially followed by reduced renin release is an important mechanism contributing to its antihypertensive effects.

5.3 EXAMPLE 3 Treatment of Hypertension Using Antisense Compounds

5.3.1 Materials and Methods

5.3.1.1 Antisense Design and Administration

AS-ODN and inverted-ODN control were 15 mer and targeted to the AUG start codon of rat β₁-adrenoceptor mRNA (Machida et al., 1990). The sequence of AS-ODN was 5′-CCGCGCCCATGCCGA-3′ (SEQ ID NO:1), and the sequence of the inverted-ODN was 5′-AGCCGTACCCGCGCC-3′ (SEQ ID NO:138). This AS-ODN was chosen from six AS candidates targeted to different regions of β₁-adrenoceptor mRNA, based on the intensity of cardiac β₁-AR inhibition and reduction of blood pressure in SHR. These oligonucleotides were modified by backbone phosphorothioation. ODNs delivered with cationic liposomes were injected into tongue vein.

5.3.1.2 Preparation of Liposomes and ODN/Liposome Complex

The cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) was mixed with a helper lipid L-α dioleoyl phosphatidylethanolamine (DOPE, Avanti Polar Lipids) at 1:1 mole ratio, briefly sonicated and stored at 4° C. until use. The average diameter of liposome is 200-350 nm (Tang and Hughes, 1998). ODN/liposome complex was prepared on the day of use by mixing desired amounts of ODNs with DOTAP/DOPE to the final DNA concentration of 300 μg/ml in 5% (wt./vol.) dextrose in water and incubating at room temperature for 60 min. Two DNA/lipid mole ratios, i.e., 1:0.5 and 1:2.5, were used in each study.

5.3.1.3 Animal Surgery

Adult male SHRs (250-350 g, Harlan) were kept in cages in a room with a 12-hr light-dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum.

5.3.1.4 Telemetric Sensor Implanation

Before implantation, the zero of each radiotransmitter (TA11PA-C40, Data Sciences) was verified to be ≦4 mm Hg. SHR were anesthetized with 100 mg/kg ketamine and 15 mg/kg xylazine and a midline abdominal incision was made. A fluid-filled catheter was then inserted into the right femoral artery and the tip of the catheter was in the abdominal aorta caudal to the renal arteries. The implanted rats were allowed to recover for one week.

5.3.1.5 Jugular Vein Cannulation

One week after telemetric implantation, rats were anesthetized and a curved catheter made of PE 50 and vinyl tubing was inserted into the external branch of the jugular vein. The tubing was led under the skin of the neck and exposed on the back to allow for drug infusion. Rats were allowed to recover for 24 hr before experimentation. The catheters were flushed with 100 U heparin every day to prevent clogging.

5.3.1.6 Membrane Preparation and β-AR Binding Assay

Four days after intravenous injection of saline (n=6) or 1 mg/kg inverted ODN (n=6), or 2, 4, 10, 18 days after injection of 1 mg/kg β₁-AS-ODN (n=24), animals were sacrificed and membranes were prepared from heart ventricles as previously described (Baker and Pitha, 1982). For saturation studies, 100 μg membrane protein was incubated in triplicate with six concentrations of ¹²⁵I-(−)iodocyanopindolol (I-CYP, NEN Life Science, 6.25-100 pM) in a total volume of 250 μl containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂ at 36° C. for 60 min. The nonspecific and β₂-adrenoceptor binding levels were determined in the presence of 1 μM (±)-alprenolol and 150 nM CGP20712A (RBI), respectively. Then the reaction mixture was passed through Whatman GF/B glass fiber filter using Brandel harvester and the bound radioactivity was counted for one min.

5.3.1.7 Tissue Preparation and Quantitative Autoradiography

Four days after injection of 1 mg/kg β₁-AS-ODN (n=6) or saline (n=6), rats were sacrificed and tissues were removed and frozen in dry ice. Coronal sections of brain, horizontal sections of heart and sagittal sections of kidney (20 μm) were cut on a cryostat (Microm) at −20° C. and mounted on microscope slides. Every seventh slide was stained with haematoxylin and eosin for histology. Tissue sections were preincubated in Krebs buffer (NaCl 118.4 mM, KCl 4.7 mM, MgSO₄ 1.2 mM, CaCl₂ 1.27 mM, NaH₂PO₄ 10.0 mM, pH 7.1) containing 0.1 mM guanosine triphosphate (GTP), 0.1 mM ascorbic acid and 10 μM phenylmethylsulfonylfluoride (PMSF) for 30 min at 25° C. Sections were then incubated in Krebs buffer containing 0.1 mM ascorbic acid and 10 μM PMSF with 100 pM I-CYP at 25° C. for 150 min, in the presence of 1 μM (−)propranolol, 100 nM IC1118,551 (β₂-selective antagonist) or 100 nM CGP20712A (β₁-selective antagonist) to distinguish non-specific, β₁- and β₂-bindings. Labeled sections were rinsed in the same buffer, followed by two 15-min washes at 37° C. in the buffer and rinsed in distilled water at 25° C. (Matthews et al., 1994). Dried sections were then exposed to X-ray films (Kodak Biomax-MR). The images were quantitated with a computerized image analysis system (MCID, Imaging Research) and normalized using ¹²⁵I-standards. Non-specific binding was less than 10% of total binding.

5.3.1.8 Determination of Effects of β₁-AS and Atenolol on Cardiovascular Parameters in Response to β-STIMULATION

5.3.1.8.1 Langendorff Heart Perfusion

Forty-eight hr after injection of 1 mg/kg β₁-AS-ODN (n=9) or inverted-ODN (n=6), SHR were anesthetized and sacrificed. Hearts were quickly removed and perfused via the aorta with oxygenated Krebs buffer (118 mM NaCl, 18.75 mM NaHCO₃, 1.2 mM KH₂PO₄, 4.7 mM KCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 11.1 mM Glucose, 0.01 mM EDTA) at a constant flow of 7.0 ml/min at 36° C. Coronary perfusion pressure (CPP) was measured via a catheter placed proximal to the aorta and connected to a pressure transducer (Gould Statham P23ID). A latex balloon filled with water and connected to the pressure transducer was inserted into the left ventricle through the left atrium to measure left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), and developed left ventricular pressure (dLVP) (dLVP=LVSP−LVEDP). LVEDP during equilibration was set at 5 to 7 mmHg. CPP, LVEDP and LVSP were continuously recorded on a 4-channel recorder (Astro-Med). After baselines for dLVP and heart rate (HR) were stable for 5 min, isoproterenol (ISO, non-specific β-agonist) was given at 0.01, 0.025, 0.05, 0.12 μM at 10-min intervals so as to avoid the effect of tachyphylaxis.

5.3.1.8.2 Telemetric Monitoring of Live Animals

The effects of β₁-AS-ODN and atenolol on cardiac dP/dt_(max), HR and systolic blood pressure (SBP) were compared in the same group of SHR (n=4). Two days after catheterization of jugular vein, control values were taken and 1 mg/kg β₁-AS-ODN was injected. Forty-eight hr later, rats were tested for the effect of β₁-AS-ODN. The rats were allowed to recover until all the cardiovascular parameters returned to control values. Then 1 mg/kg atenolol (β₁-selective antagonist) was injected and rats were tested 30 min later. For β₁-stimulation, SHR were infused with dobutamine (β₁-selective agonist) through jugular vein catheter at 5, 10, 20, 40 μg/kg/min. Each dose was given for 5 min continuously and at 1-hr intervals until all cardiovascular parameters returned to baseline so as to avoid the effect of tachyphylaxis. BP and HR were sampled every min. dP/dt_(max) was calculated from the slope of the rising pulse pressure curve and determined every min. The difference between values at each dose and baseline was denoted as Δ.

5.3.1.9 Blood Pressure Monitoring

5.3.1.9.1 Telemetry

Each rat cage was placed on a receiver (RLA1020, Data Sciences) for measurement of cardiovascular parameters. Data were collected with a computer-based data acquisition program (Dataquest LabPRO3.0; Data Sciences). BP and HR were measured every 10 min and averaged every 1 to 24 hr. Before treatment, SHR were monitored for a week to get a stable baseline.

5.3.1.9.2 Tailcuff

Rats were warmed for 20-30 min in cages on heating pads. The temperature was controlled at 35-37° C. Then rats were placed in a plastic restrainer kept at 37° C. A pneumatic pulse sensor was attached to the tail. After cuff inflation, SBP was determined as the first pulsatile oscillation on the descending side of pressure curve. HR was determined by manual counting of pulse numbers per unit time. BP and HR were recorded by a Narco physiograph. Data values of each rat were taken as an average of at least four stable readings. Baseline was determined by averaging three days of measurements before antisense administration.

5.3.1.10 Statistic Analysis

Values were expressed as mean±SEM. Difference was considered statistically significant at P<0.05. Unpaired t-test was used to compare B_(max), dLVP and BP in two groups. One way repeated ANOVA and Tukey test were used to compare ΔdP/dt and ΔHR upon dobutamine infusion in different groups. Pearson product moment correlation was used to assess the relationship between β₁-adrenoceptor B_(max) and dLVP.

5.3.2 Results

5.3.2.1 Effect on Cardiac β-Adrenoceptor Density

A single intravenous injection of 1 mg/kg β₁-AS-ODN delivered with cationic liposomes at a 1:2.5 mole ratio significantly reduced the β₁-adrenoceptor densities in the SHR hearts for 18 days (P<0.01). The drop was at its maximum of 47% on day 4, 33% on day 10, and maintained at 29% on day 18. In contrast, there was no significant change in the B_(max) of β₂-adrenoceptors. K_(D) of both subtypes remained unaltered (Table 5). Consequently, the β₁/β₂ subtype ratio in the ventricles was diminished from ˜70/30 to ˜50/50 by β₁-AS-ODN. Inverted-ODN had no effect on either subtype.

TABLE 5 B_(MAX) AND K_(D) OF β₁- AND β₂-ADRENOCEPTORS IN CARDIAC VENTRICLES OF SHR 4 DAYS AFTER TREATMENT WITH SALINE, INVERTED-ODN OR β₁-AS-ODN Total (β₁ + β₂) β₁ β₂ K_(D) B_(max) K_(D) B_(max) K_(D) B_(max) (pM) (fmol/mg) (pM) (fmol/mg) (pM) (fmol/mg) Saline 55.4 ± 6.7 27.6 ± 0.9 64.7 ± 5.4 18.7 ± 0.9 29.6 ± 0.5 9.1 ± 0.6 Inverted- 57.1 ± 3.5 26.3 ± 3.0 66.0 ± 1.0 18.1 ± 2.3 32.0 ± 2.4 8.7 ± 0.5 ODN β₁-AS- 37.0 ± 1.0* 19.8 ± 1.8* 60.5 ± 3.4* 10.0 ± 1.2* 30.7 ± 3.3 9.9 ± 0.5 ODN Data represent mean ± SEM of each group (N = 6-10). *P < 0.01 versus saline control.

5.3.2.2 Effect on Cardiac Contractility and Heart Rate in Response to β-Stimulation

Forty-eight hr after injection of 1 mg/kg β₁-AS-ODN, the cardiac inotropic and chronotropic responses to p-stimulation were determined in SHR in vitro and in vivo.

First, isolated hearts were perfused with incrementing doses of ISO, which enhanced heart rate and contractility via activating β-adrenoceptors. The dLVP-ISO dose-response curve, which reflected the positive inotropic effect of ISO, was significantly shifted down by β₁-AS-ODN (P<0.02). Heart rate was not significantly decreased.

In vivo test was performed in conscious SHR monitored by radiotelemetry. β₁-AS-ODN significantly (P<0.02) dampened the increase in dP/dt_(max), in the face of dobutamine (β₁-selective agonist). The change in heart rate was not significantly reduced, which echoed the results in isolated, perfused hearts. On the other hand, the response of blood pressure to dobutamine was biphasic. Dobutamine elevated SBP by 5-8 mmHg at low infusion speed and reduced SPB at higher speed. This is probably due to the partial β₂-argonistic activity of dobutamine at high doses and the consequent vasodilatory effect on blood pressure. Relative to β₁-AS-ODN, 1 mg/kg atenolol produced a more profound decline in ΔdP/dt_(max) and ΔHR during a 5-hr period of time after injection. Moreover, it reduced the resting dP/dt_(max) (2450±295 mmHg/sec versus control 2937±277 mmHg/sec) and caused bradycardia (295±12 bpm versus control 365±8 bpm) (P<0.05), while β₁-AS did not change resting contractility (2922±249 mmHg/sec) or heart rate (365±12 bpm). But the effects of atenolol on ΔdP/dt_(max) and ΔHR were transient. Within 24 hr after atenolol administration, the inotropic and chronotropic effects of dobutamine had returned to control level.

5.3.2.3 Effect on blood Pressure of SHR

The effects of single injection of 1 mg/kg β₁-AS-ODN on blood pressure of SHR measured by tailcuff method were observed. AS-ODN was delivered with cationic liposome at different mole ratios of DNA/lipid, ie., 1:0.5 and 1:2.5. β₁-AS-ODN delivered with liposomes at 1:0.5 ratio diminished SBP for 8 days. The maximum drop was 38±5 mmHg. When the mole ratio was increased to 1:2.5, this hypotensive effect was drastically prolonged to 20 days. No effect was seen with inverted-ODN.

In order to compare the effects of β₁-AS-ODN and atenolol, radiotelemetry was used to monitor blood pressure and heart rate on a regular basis. One mg/kg β₁-AS-ODN delivered with liposomes at a 1:0.5 ratio produced a maximum drop of 15 mmHg in mean blood pressure. The antihypertensive effect lasted for 8 days, which was consistent with the results measured with tailcuff. Heart rate was not significantly altered. In contrast to AS-ODN, although the onset of hypotensive effect caused by 1 mg/kg atenolol occurred as early as 20 min after injection, it lasted for only 10 hr. In addition, atenolol caused considerable bradycardia up to an average of −75 bpm.

5.3.2.4 Effect on β-Adrenoceptor Distribution in Brain, Heart and Kidney

Quantitative autoradiography of 6-18 tissue slices in brain, heart and kidney was analyzed four days after intravenous β₁-AS-ODN administration. No changes in the distribution of β-adrenoceptors in forebrain and brainstem regions were detected. This indicated the absence of antisense effect on the β-adrenoceptor expression in CNS. However, β₁-AS-ODN significantly (P<0.05) reduced β₁-adrenoceptor densities in cardiac ventricles (from 30.2±2.1 to 20.6±2.5 fmol/mg) and renal cortex (26.4±3.1 to 17.4±3.3 fmol/mg). This was consistent with the binding results. β₂-adrenoceptors were not affected in any tissues.

5.3.3 Summary

This example demonstrates the effects of β₁-AS-ODN compositions on high blood pressure in a model of hypertension as compared to using a traditional β-blocker modality. β₁-AS-ODNs knocked down β₁-adrenergic activity, resulting in long-term attenuation of blood pressure. The results indicated that β₁-AS-ODN reduced β₁-adrenoceptor density and cardiac contractility following β₁-stimulation in vitro and in vivo and lowered high blood pressure of SHR. These findings are consistent with the hypothesis that β₁-AS, through the inhibition of β₁-adrenoceptor expression, is able to render heart, kidney and other tissues less sensitive to sympathetic activation, which is a major contributing factor in high blood pressure. A single injection of β₁-AS-ODN effectively decreased the cardiac β₁-adrenoceptor density, which was accompanied by diminished ventricular contractility and cardiac output in response to β-stimulation. The antihypertensive effect in SHR was up to a 38-mmHg reduction lasting as long as 20 days.

Treatment of β₁-AS-ODN reduced cardiac β₁-adrenoceptor density by ˜50% in four days. There is considerable variation reported in the literature on the half-life of β₁-adrenoceptors (Baker and Pitha, 1982; Neve and Molinoff, 1986; Winter et al., 1988). From the results with the AS-ODN inhibition, it appears that the half-life of cardiac β₁-adrenoceptor is approximately 2-4 days to allow for 50% reduction of β-adrenoceptor density within 4 days.

Both tailcuff and telemetry measures of blood pressure showed a significant drop after antisense treatment. SHR responded to β₁-AS-ODN to a greater degree of hypotension when subjected to tailcuff versus telemetry. In addition, the baseline measured with tailcuff was consistently higher than that with telemetry by 20-30 mmHg in the same rats. Bazil et al. (1993) reported a similar phenomenon. They compared the cardiovascular parameters recorded by telemetry, tailcuff and arterial catheter and observed a more sensitive hypotensive effect of captopril with tailcuff. Tailcuff measurement of blood pressure involves warming and restraint of rats. It is conceivable that β₁-AS, through the suppression of sympathetic activity, can decrease blood pressure of animals under stress more effectively.

Cationic liposomes are effective vehicles for gene delivery. It has been shown that liposome entrapment not only improves the cellular uptake of DNA, but also protects DNA from degradation and extends its circulation time (Allen, 1997; Liu et al., 1997). Numerous factors influence the efficiency of cationic liposome-mediated intravenous gene delivery, such as DNA/lipid ratio, selection of lipids and preparation procedure (Liu et al., 1997; Yang and Huang, 1998). A widely used lipid formula DOTAP/DOPE was used to deliver β₁-AS-ODN at four mole ratios (1:0.5, 1:1.5, 1:2.5, and 1:3.5). The optimal ratio 1:2.5 was determined based on the duration and magnitude of the antihypertensive effects. A profound and prolonged fall in blood pressure of 38 mmHg up to 20 days was achieved at this ratio, which followed the reduction in β₁-adrenoceptor binding to a maximum of 47% at day 4, 33% at day 10, and 29% at day 18. This implies that β₁-AS-ODN effectively inhibits the functionally active receptors involved in the blood pressure.

The blood-brain barrier formed by capillary endothelia is only permeable to small lipophilic molecules with molecular weight <600 Da (Padridge, 1998). Owing to their high hydrophilicity, ODNs undergo negligible transport through blood-brain barrier and have very limited access to CNS (Agrawal et al., 1991). The cellular and organ distributions of DNA/liposome complexes with fluorescent labeling were previously studied in mice after intravenous injection and the results indicated that the complexes were primarily taken up by capillary endothelial cells in most of the peripheral organs including lung, heart, kidney and spleen, but absent in the brain (McLean et al., 1997). Although the brain retention of liposomes after peripheral administration was observed in some cases, it is due to entrapment within the brain microvasculature (Schackert et al., 1989). In this study, autoradiography in brain revealed no detectable changes in the expression and distribution of β-adrenoceptors after intravenous β₁-AS-ODN injection. This provided further evidence that an antisense modality does not produce CNS effects.

High specificity based on gene sequence has made antisense an increasingly useful tool in numerous studies and clinical trials. Its success is manifested by the recent approval of the first antisense drug Vitrovene by FDA. In the present study, β₁-AS-ODN inhibited β₁-AR expression without changing β₂-adrenoceptors, demonstrating a specificity of the β₁-AS-ODN selected sequences.

In patients with chronic heart failure, the severity of the disease closely relates to the decrease in cardiac β-AR density and functional responsiveness (Brown et al., 1992). In SHR treated with β₁-AS-ODN, a marked attenuation of the β₁-adrenoceptor-mediated positive inotropic response was observed in vitro and in vivo, concurrent with the diminished cardiac β₁-AR level. Therefore, these results indicated a positive correlation between cardiac β₁-AR number and functional sensitivity (correlation coefficiency >0.90, P<0.01).

Despite the large decrease in blood pressure, no reflex tachycardia was observed after antisense treatment. However, the suppressive effect of β₁-AS-ODN on heart rate was less significant than its negative inotropic response. The reason for this is still under investigation. Several possibilities can be considered. First, β₁-AS-ODN did not affect β₂-adrenoceptors, which played an important role in the regulation of heart rate (Kaumann, 1986; Rodefeld et al., 1996), while not involved in the cardiac contraction. Second, antisense inhibition of β₁-AR expression is gradual and to a lesser extent than β-blockers. It is also possible that β₁-ARs may have a larger reserve for controlling heart rate than contractility. Finally, evidence suggests that cardiomyocytes preferentially take up AS-ODN while pacemaker cells are less efficient.

The cardiovascular effects of β₁-AS-ODN were compared with a hydrophilic β₁-selective antagonist, atenolol. β₁-AS-ODN showed advantages over atenolol in reducing blood pressure and maintaining normal heart rate. Although the onset of β₁-AS-ODN action was slower than atenolol, it lasted much longer, 20 days compared to less than 1 day with atenolol. Furthermore, β₁-AS-ODN did not affect heart. rate, while atenolol caused appreciable bradycardia. Bradycardia is a common complaint by patients taking this drug. Atenolol also reduced resting ventricular contractility and heart rate and thereby reduced resting cardiac output. β₁-AS-ODN is unlikely to alter resting cardiac performance. The results presented here suggest that β₁-AS-ODN offer a significant improvement over currently used β-blockers, in both prolonged blood pressure reduction and absence of effects on β₂-adrenoceptors and CNS.

5.4 Example 4 Protection Against Myocardial Ischemia-Reperfusion-Induced Cardiac Dysfunction by Antisense Compositions Directed at β₁-AR mRNA

Acute myocardial ischemia causes significant increase in plasma catecholamine levels, which leads to exacerbation of the ischemic myocardial injury (Waldenstrom et al., 1978; Rona, 1985). The worsening myocardial ischemia is an important factor in cardiac dysfunction. Acute myocardial ischemia is also characterized by increased sensitivity of β₁-ARs in the myocardium during acute ischemia (Strassere et al., 1990). β-ARs form the interface between the sympathetic nervous system and the cardiovascular system (Strassere et al., 1990; Mukherjee et al., 1979; Maisel et al., 1985. Importantly, β₁-AR subtype 1 (β₁-AR) is by far the predominant β-AR in myocardium (Minneman et al., 1995) and its activity and sensitivity are believed to regulate cardiac function via adenylyl cyclase activity (Mukherjee et al., 1979; Thandroyen et al., 1986; Böhm, 1995). Several studies have shown that density of β₁-AR increases and the expression of β₁-AR mRNA is augmented in the myocardium after acute ischemia (Maisel et al., 1985; Karliner et al., 1989; Ihl-Vahl et al., 1995). Experimental and clinical results have also demonstrated that β-AR blockade, especially selective blockade of β₁-AR, can protect myocardium against ischemic injury and cardiac dysfunction (Schulz et al., 1995; Ablad et al., 1987; Lu et al, 1990), decrease infarct size (Schulz et al., 1995), and reduce the incidence of sudden cardiac death in patients with myocardial infarction (Yusuf et al., 1985).

Although chemical β-AR blockers are commonly used in the treatment of ischemic heart disease, these agents often cause central nervous system side effects and β₂-AR antagonistic activity is associated with increase in peripheral vascular resistance. Development of antisense-oligodeoxynucleotides (AS-ODNs) against specific receptor mRNA is a novel approach to decrease the synthesis of receptor proteins (Phillips et al., 1996; Dachs et al., 1997). This approach has potential to be of therapeutic benefit in disease states characterized by upregulation of these receptors (Phillips et al., 1996; Dachs et al., 1997; Yang et al., 1998). A recent study indicated that AS-ODNs directed at angiotensin II type 1 receptors (AT₁) decrease the synthesis of AT₁ receptor protein and protects the ischemic rat heart from the adverse effects of ischemia (Yang et al., 1998). As demonstrated herein, a single intravenous injection of an antisense compositions directed against β₁-AR mRNA can reduce blood pressure in SHR for periods of at least 20 days (Zhang et al., 1999).

In this example, the effect of AS-ODNs on lipid peroxidation was examined. Also examined were their effects on the expression of β₁-AR protein and mRNA in the myocardium after ischemia-reperfusion.

5.4.1 Materials and Methods

5.4.1.1 Antisense Compositions and Liposomal Formulations

AS-ODNs and inverted-oligodeoxynucleotides (IN-ODNs) control were 15-mers and targeted to −5 to +10 of rat β₁-AR mRNA encompassing the AUG start colon. The sequence of AS-ODNs was 5′-CCGCGCCCATGCCGA-3′ (SEQ ID NO:1), and the corresponding IN-ODNs was 5′-AGCCGTACCCGCGCC-3′ (SEQ ID NO:138) (Zhang et al., 1999). These ODNs were modified by backbone phosphorothioation and synthesized in the DNA Synthesis Core Laboratory of the University of Florida.

Since cationic liposomes enhance the uptake of DNA by cells and also protect DNA from degradation and extend its circulation time, DOTAP/DOPE (mole:mole=1:1) liposomes were used to deliver the antisense compositions to host animals. The average diameter of liposomes was 200-300 nm. ODNs/liposomes complex was prepared on the day of use by mixing desired amount of ODNs with DOTAP/DOPE to final DNA concentration of 300 μg/ml in 5% (wt./wt.) dextrose in water and incubating at room temperature for 60 min (Yang et al., 1998; Zhang et al., 1999).

5.4.1.2 Animals

Male Sprague-Dawley rats weighing 200-250 g were injected intravenously with either AS-ODNs (n=7) or IN-ODNs (n=7) at a dose of 200 μg/rat 4 days before excising the hearts. DOTAP/DOPE liposomes (700 μg/rat) were given along with ODNs. Parallel groups of rats were treated with saline (n=13), or the selective β₁-AR blocker atenolol 2 mg/kg (n=7) 6 hr before the hearts were excised.

5.4.1.3 Isolated Perfused Heart Model

Four days after administration of AS-ODNs or IN-ODNs or 6 hr after administration of atenolol or saline, rats were anesthetized with sodium pentobarbital (40 mg/kg) intraperioneally. The hearts were excised rapidly and placed in ice-cold Kreb-Henseleit buffer (mmol/L: NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.25, NaHCO₃ 25, and glucose 11, pH 7.4). Within one min, the hearts were transferred to an isolated perfusion apparatus and perfused via the aorta with oxygen-saturated (95% O₂+5% CO₂) Kreb-Henseleit buffer kept at 37° C. with the use of a MasterFlex pump (model 7015-21, Cole-Palmer Instrument Co.) according to the modified Langendroff procedure (Yang et al., 1998; Neely and Rovetto, 1975). The heart was placed in a semi-closed circulating water-warmed (37° C.) air chamber, paced atrially with a Medtronic 5320 pacemaker at a rate of 300 bpm, and perfused at a constant flow (5.5-6.0 ml/per min). Coronary perfusion pressure (CPP) was measured via a catheter placed just proximal to the aorta and connected to a Gould Statham P23ID pressure transducer. A latex balloon filled with water and connected to a Gould Statham P23ID pressure transducer was inserted in the left ventricle through the left atrium to measure left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), and developed left ventricular pressure (dLVP) (dLVP=LVSP−LVEDP). LVEDP during equilibration was set at 5 to 7 mmHg. All measurements were continuously recorded on a 4-channel record (Astro-Med).

5.4.1.4 Myocardial Ischemia and Reperfusion

Six hearts from saline-treated rats were continuously perfused with Kreb-Henseleit buffer for 80 min and served as sham control. Hearts from other rats, after 20 min of equilibration, were subjected to 30 min of ischemia followed by 30 min of reperfusion. After completion of the study, hearts were frozen in liquid-nitrogen for β₁-AR analysis by binding assay, β₁-AR protein analyses by Western blot, β₁-AR mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR™), and measurement of MDA.

5.4.1.5 Determination of β₁-AR Density in Myocardium

Membrane protein was prepared from left ventricles as previously described (Baker and Pitha, 1982). For saturation studies, 100 μg membrane protein was incubated in triplicate with ¹²⁵I-(−)iodocyanopindolol (I-CYP, NEN Life Science, 6.25-100 pM) in a total volume of 250 μl containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂ at 36° C. for 60 min. The nonspecific and β₂-adrenoceptor binding were determined in the presence of 1 μM (±)-alprenolol and 150 mM CGP207ASA (RBI), respectively. Then the reaction mixture was passed through Whatman GF/B glass filter using Brandel harvester and the radioactivity was counted for one min.

5.4.1.6 Quantification of β₁-AR Protein Expression in Myocardium

Myocardial tissues were homogenized and lysed in boiling lysis buffer (1% SDS, 0.1% Triton X-100®, and 10 mmol/L Tris-HCl, pH 7.4) and centrifuged at 10,000 RPM for 30 minutes at 4° C. The lysate protein from myocardial tissues (20 μg/lane) was separated by 8% SDS-PAGE using a Bio-Rad Mini-Protean cell, transferred to nitrocellulose membrane (Amersham). After incubation in blocking solution (4% non-fat milk, Sigma), membranes were incubated with 1:1000 dilation primary antibody (polycolonal antibody to β₁-AR, Santa Cruz Biotechnology) for overnight at 4° C. Membranes were washed and incubated with 1:2000 dilution second antibody (Amersham) for one hr. The membranes were detected with the ECL system, as described previously (Yang et al, 1998; Li et al., 1999).

5.4.1.7 Determination of β₁-AR mRNA

Total RNA was isolated from rat myocardium with the single step acid-guanidinum thiocyanate-phenol-chloroform method and quantified (Chomcznski and Sacchi, 1987). One μg of total RNA was reverse transcribed with oligo-dT (Promega) and M-MLV reverse transcriptase (Promega) at 37° C. for one hr. 1.5 μl of RT material was amplified with Taq DNA polymerase (Promega) using a primer pair specific to β₁-AR:

Forward primer: 5′-CTCCGAAGCTCGGCATGG-3′ (SEQ ID NO:139); and

Reverse primer: 5′-GCACGTCTACCGAAGTCCAGA-3′ (SEQ ID NO:140).

PCR™ product was 432 base pairs. For PCR, 35 cycles were used at 95° C. for one minute, 60° C. for one minute, and 72° C. for one min. The RT-PCR™ amplified samples were visualized on 1.8% agarose gels using ethidium bromide. A primer pair of rat GAPDH was used as control:

Forward primer: 5′-ATCAAATGGGGTGCTGGTGCTG-3′ (SEQ ID NO:141); and

Reverse primer: 5′-CAGGTTTCTCCAGGCGGCATGTCA-3′ (SEQ ID NO:142).

For PCR™, 35 cycles were used at 95° C. for one min, 60° C. for one min, 72° C. for one min. PCR™ product was 504 base pairs. Relative intensity of bands of interest were analyzed by NSF-300G Scanner (Microtek) (Yang et al., 1998; Li et al., 1999).

5.4.1.8 Determination of MDA Levels in Myocardium

Malondialdehyde (MDA) levels in myocardium were measured in duplicate by a modification of the method of Ohkawa et al., (1979). Briefly, the ventricular tissues were homogenized. The assay mixture consisted of 0.1 ml of the tissue homogenate, 0.4 ml of 0.9% NaCl, 0.5 ml of 3% sodium dodecylsulfate, 3 ml of TBA (thiobarbituric acid reagent, containing equal parts of 0.8% aqueous thiobarbituric acid and acetic acid) and was heated for 75 min at 95° C. Thereafter, 1 ml cold 0.9% NaCl was added to the mixture, which was cooled and extracted with 5 ml n-butanol. After centrifugation at 3,000 rpm for 15 min, the butanol phase was assayed spectrophotometrically at 532 nm. Tetramethoxypropane (in amounts of 0, 0.1, 0.2, 0.4, 0.8, 1.0 nmole) served as external standard. MDA levels in myocardium were expressed in μmol/g tissue.

5.4.1.9 Data Analysis

Data was presented as mean±SD. Statistical significance was determined in multiple comparisons among independent groups of data in which ANOVA and the Student-Newman-Keuls test indicated the presence of significant differences. A P value of ≦0.05 was considered statistically significant.

5.4.2 Results

5.4.2.1 Cardiac Dysfunction During Ischemia-Reperfusion

The basal values of CPP, LVSP, LVEDP, and dLVP were similar in all groups of rat hearts. In the control continuously buffer-perfused hearts observed for 80 min, there were only minimal (˜5%) changes in the indexes of cardiac fiction. In the hearts from saline-treated rats, 30 min of ischemia followed by 30 min of reperfusion resulted in marked cardiac dysfunction, indicated by a significant increase in CPP and LVEDP, and a decrease in LVSP and dLVP (all P<0.01, vs. pre-ischemia values).

Treatment of rats with AS-ODNs markedly attenuated the ischemia-reperfusion-induced myocardial dysfunction, indicated by preservation of LVSP and dLVP and minimization of increase in LVEDP and CPP (all P<0.01, vs. saline group). Treatment of rats with atenolol also reduced the increase in CPP and LVEDP induced by ischemia-reperfusion (all P<0.05, vs. saline group, n=7), and modestly attenuated the ischemia-reperfusion-induced change in LVSP and dLVP (P<0.05 vs. saline group, n=7). AS-ODN treatment was more effective than atenolol treatment in preserving dLVP after ischemia-reperfusion (P<0.05). Treatment with IN-ODNs showed no effect on ischemia-reperfusion-induced myocardial dysfunction.

5.4.2.2 MDA Levels in Myocardium

MDA levels in myocardium increased significantly after ischemia-reperfuision (P<0.05 vs. sham control hearts, n=6). Pretreatment of rats with AS-ODNs and atenolol attenuated the increase in MDA levels in the myocardium (both P<0.05 versus saline pretreatment, n=7 each group). As expected, IN-ODNs did not affect MDA levels in myocardium.

5.4.2.3 Change of β₁-AR Density in Myocardium After Ischemia-Reperfusion

Myocardium from sham control continuously perfused hearts continuously exhibited β₁-AR and β₂-AR (β₁-AR>>β₂-AR). After ischemia-reperfusion in the saline-treated rat hearts, there was a consistent increase in β₁-AR density in myocardium (B_(max) 28.7±5.4 vs. 19.6±1.7 fmol/mg in continuously perfused buffer-perfused rat hearts, P<0.05, n=4), while there was no change in β₂-AR density. Treatment of rats with AS-ODNs resulted in a decrease in β₁-AR density in the ischemic-reperfused myocardium (B_(max) 15.9±1.3 vs. 28.7±5.4 fmol/mg in saline-treated rat hearts, P<0.05, n=4) while there was no change in β₂-AR density. Treatment of rats with INV-ODNs or atenolol had no effect on β₁-AR density in the ischemic-reperfused myocardium.

5.4.2.4 Express of β₁-AR Protein and mRNA in Myocardium

Western analysis of the control continuously perfused hearts showed a distinct β₁-AR protein band of 41 kDa. Similar molecular weight band was observed in hearts from saline-, AS-ODNs-, IN-ODNs- and atenolol-treated rat hearts. The β₁-AR protein band was very dense in the saline-treated rat hearts, including upregulation of the protein during ischemia-reperfusion. Treatment of rats with AS-ODNs abolished the ischemia-reperfusion-mediated increase in β₁-AR protein expression. Notably, treatment of rats with IN-ODNs or atenolol had no effect on the density of β₁-AR protein.

Ischemia-reperfusion also resulted in an increase of mRNA for β₁-AR signal (adjusted for GAPDH signal) by myocardium of saline-treated rats, as determined by RT-PCR. Pretreatment of rats with AS-ODNs attenuated the increase of mRNA for β₁-AR in myocardium, but mRNA level for β₁-AR in the myocardium was not significantly affected by treatment of rats with IN-ODNs or atenolol.

5.4.3 Summary

The present example demonstrates the protective role of AS-ODNs directed at β₁-AR mRNA against cardiac dysfunction after a brief period of ischemia-reperfusion. The study compared the effects of pretreatment of rats with AS-ODNs directed at β₁-AR mRNA or a selective β₁-AR blocker atenolol in this process. This study showed that ischemia for 30 min followed by reperfusion for 30 min resulted in a significant cardiac dysfunction and lipid peroxidation in saline-treated rat hearts. Further, ischemia-reperfusion was associated with a marked upregulation of β₁-AR density and protein and mRNA expression. Pretreatment of rats with atenolol preserved cardiac function, but did not affect the density of β₁-AR and the expression of β₁-AR protein and mRNA in the ischemic-reperfused myocardium. Pretreatment of rats with AS-ODNs directed at β₁-AR mRNA provided almost total preservation of cardiac function and lipid peroxidation following ischemia-reperfusion. AS-ODN treatment also prevented the upregulation of β₁-AR density, protein and mRNA expression in the ischemic-reperfused myocardium. The effects of AS-ODNs directed at β₁-AR mRNA on cardiac function were clearly superior to those of the commonly used β₁-AR blocker atenolol. The effects of AS-ODN on β₁-AR protein and mRNA expression indicate that its beneficial effects are mediated by inhibition of both transcription and translation of β₁-AR mRNA.

There is a generalized stimulation of the sympathetic nervous system during ischemia, perhaps a compensatory response designed to preserve cardiac dysfunction. Accordingly, catecholamine levels increase in both plasma and myocardium following myocardial ischemia (Rona, 1985; Richardt et al., 1994; Abrahamsson et al., 1983), but the increased catecholamine concentrations have the potential to contribute to increased excitability of myocardiun resulting in arrhythmia (Strassere et al., 1990; Mukherjee et al., 1979; Maisel et al., 1985; Ohyanahi et al., 1988; Maisel et al., 1987). There is also increase in sensitivity of β₁-AR during acute myocardial ischemia (Strassere et al., 1990). This coupled with increased circulating and myocardial catecholamine concentrations can exacerbate cardiac injury and dysfunction.

It is generally accepted that activation of β-AR is the first element in the signal transduction chain mediating sympathetic stimulation of the heart. β₁-ARs, the dominant β-AR subtype in the heart (Minneman et al., 1995; Guderman et al., 1995), regulate cardiac function by activating adenylyl cyclase activity (Strassere et al., 1990; Thandroyen et al., 1986; Bohm, 1995). Several experimental studies have indicated an upregulation in β₁-ARs, but not β₂-ARs, during acute ischemia-reperfusion (Maisel et al., 1985; Karliner et al., 1989; Persad et al., 1998). Ihl-Vahl et al. (1995) conclusively demonstrated a rapid upregulation of β-AR mRNA during acute myocardial ischemia; this upregulation is subtype-selective with a specific increase mRNA level for β₁-ARs, but not for β₂-ARs. They also showed that the increase of β₁-AR mRNA is ischemia time-dependent.

Pretreatment of animals with β₁-AR blockers does not affect the ischemia-reperfusion-induced increase in β₁-AR mRNA level. This was confirmed in the present study wherein treatment with atenolol did not affect either the β₁-AR density measured by binding assays or the augmented β₁-AR protein and mRNA levels measured by Western analysis and RT-PCR, respectively. Other investigators have also shown that β₁-AR blockers do not block the expression of β₁-AR protein or mRNA (Aarons and Molinoff, 1982; Heilbrunn et al., 1986; Aarons et al., 1980). In contrast, the present study clearly demonstrates that AS-ODNs directed at β₁-AR mRNA block the up-regulation of β₁-AR number, and protein and mRNA levels. The blockade of β₁-AR at the transcriptional level may be the basis for the superior effect of the AS-ODN approach in the preservation of cardiac function after acute ischemia-reperfusion. Therapy with a single dose of AS-ODNs four days prior to removal of hearts prevented the increase in β₁-AR protein, which implies sustained inhibitory effects of AS-ODNs at translational levels as well. A previous study in the SHR indeed demonstrated that AS-ODNs against β₁-AR mRNA decreases β₁-AR mRNA translation into protein in the hearts for at least up to 18 days (Zhang et al., 1999).

Although chemical β-AR blockers are effective in the therapy of ischemic heart disease and are widely used in the short- and long-term management of patients with myocardial ischemia, these agents have several undesirable side-effects related to their effects on central nervous system, peripheral vascular resistance, and tracheo-bronchial tree. Even the selective β₁-AR blockers, such as atenolol, lose their cardio-selectivity at moderate doses. In addition, these agents need to be taken frequently, at least one daily, due to their short half-life. Furthermore, chemical β-AR blockers do not influence β-ARs at genomic level. Gene therapy, such as AS-ODNs directed at β₁-AR mRNA, provides unique benefits. For example, the sequence specific AS-ODNs directed at β₁-AR mRNA can significantly decrease ventricular β₁-AR density by 30-50% even after 18 days in the rat after a single intravenous injection (Zhang et al., 1999). The AS-ODNs has no effect on β₂-AR density. Concurrently, AS-ODN administration is associated with reduction in blood pressure with a 38-mmHg maximum drop without causing bradycardia. Most importantly, there is no effect of AS-ODNs on the distribution of β-ARs in brain. These results demonstrate that a single intravenous injection of AS-ODNs can preserve cardiac dysfunction and protect myocardial injury from ischemia-reperfusion in the rat with specific effects on the expression of mRNA for β₁-ARs in myocardium. Importantly, IN-ODNs, used as control for AS-ODNs, did not show any of these effects.

In this study, ischemia-reperfusion-induced cardiac dysfunction was evaluated by the measurement of CPP, LVEDP, LVSP and dLVP. These indices of myocardial dysfunction have been used in several studies in the isolated heart model of global ischemia-reperfusion (Yang et al., 1998; Yang et al., 1993; Yang et al., 1997; Kokita et al., 1998; Ozden et al., 1998). Isolated rat, rabbit or guinea pig heart model provides an inexpensive and reproducible method to evaluate cardiac function and myocardial metabolic alterations during ischemia-reperfusion. This model has been used extensively to study regulation of variety of receptors and modulation of cardiac function by agents acting on different receptors (Lu et al., 1990; Yang et al., 1998; Neely and Rovetto, 1975; Baker and Pitha, 1982; Maisel et al., 1987; Yang et al., 1993; Yang et al., 1997; Kokita et al., 1998; Ozden et al., 1998). In the isolated beating heart, cardiac function can be assessed independent of the influence of circulating blood cells and hormones, which may be considered to provide an important advantage of this model.

MDA, a lipid peroxidation product, has been widely accepted as an index to evaluate myocardial injury after ischemia-reperfuision (Kokita et al., 1998; Ozden et al., 1998). All these parameters were modified by the use of AS-ODNs in the present study. In summary, the present study is the first report on the amelioration of cardiac dysfunction and myocardial injury induced by ischemia-reperfusion in isolated rat heart with a single intra-venous injection of AS-ODNs directed at β₁-AR mRNA. This study also provides evidence that the AS-ODNs can block the augmented expression of mRNA for β₁-AR in the ischemic myocardium. Lastly, the effects of AS-ODNs directed against β₁-AR mRNA appear to be superior to those of atenolol in the isolated rat heart model of ischemia-reperfusion.

5.5 Example 5 Reduction of Cardiac Hypertrophy in Animal Models

Adult spontaneously hypertensive rats (300-350 g) were treated with β₁-AS-ODNs for more than 2 months. The blood pressure was reduced by 20-30 mmHg persistently throughout the treatment period (FIG. 11). Control animals receiving saline or INV-ODNs had no change in blood pressure. At the end of a 2-month treatment, the hearts were dissected and weighed. Left ventricular hypertrophy was indicated by the ratio of left ventricle weight to body weight (LV/BW). β1-AS-ODNs significantly reduced left ventricular hypertrophy (P<0.05), compared to controls (FIG. 11).

5.6 Example 6 Attenuation of Cardiac Dysfunction After Myocardial Ischemia-Reperfusion

The protective effects of β1-AS-ODNs were tested in a rat model of myocardial ischemia-reperfusion. Five groups of SD rats were studied. Sham controls (n=6) were injected with saline and perfused continuously for 80 min without ischemia/reperfusion (I/R). In other groups, hearts were perfused for 20 min and subjected to global ischemia (30 min) followed by reperfusion (30 min). Saline+I/R (n=6) or atenolol+I/R (n=7) groups were injected i.v. with saline or 2 mg/kg atenolol 6 hr before the hearts were excised. AS-ODN+I/R (n=7) or INV-ODN+I/R (n=7) were injected iv. with 1 mg/kg β₁-AS-ODN or INV-ODN 4 days before the I/R study was performed.

Ischemia-reperfusion resulted in significant cardiac dysfunction, indicated by a significant increase in coronary perfusion pressure (CPP) and left ventricular end-diastolic pressure (LVEDP), and a decrease in developed left ventricular pressure (dLVP) (P<0.01 vs. pre-ischemia values).

Treatment with β₁-AS-ODN markedly attenuated the ischemia-reperfusion induced ventricular abnormality, manifested by the preservation of dLVP and minimization of increase in CPP and LVEDP (P<0.05 vs. saline group). Overall, AS-ODN treatment appeared to be equivalent to or even better than atenolol in these effects. AS-ODN restored dLVP to a greater extent than atenolol. INV-ODN showed no effect on ischemia-induced dysfunction and cannot be differentiated from saline group. Data on cardiac function parameters from these studies are summarized in FIG. 12.

5.7 Example 7 Safety Profile of Chronic use of β₁-AS-ODNs

Safety profiles including liver transaminases, hematology, immune response and tissue pathology were investigated during three repeated injections of β₁-AS-ODNs for extended periods of time. The results indicated that long-term treatment with β₁-AS-ODNs is safe without causing toxic effects or immune reaction.

5.7.1 Liver Transaminases

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are routinely used as a clinical index for liver function. Their levels increase substantially upon liver damage. SHR were treated with three repeated injections of β₁-AS-ODN. Plasma concentrations of ALT and AST were measured after each injection, which revealed no significant difference between AS-ODN and control groups (FIG. 13).

5.7.2 Hematology

Hematological parameters were measured after three repeated injections of β₁-AS-ODN. There was no change in total leukocyte count (WBC), platelet count, hematocrit and mean platelet volume (MPV) among three groups (Table 6).

TABLE 6 CLINICAL BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS AFTER REPEATED ADMINISTRATION OF β₁-AS-ODN β1-AS-ODN INV-ODN Saline Liver Enzymes AST (unit/ml) 74.0 ± 3.7  83.0 ± 8.3 82.3 ± 3.8 ALT (unit/ml) 25.7 ± 1.1  30.5 ± 3.8 30.0 ± 3.2 Hematology hematocrit (%) 46.2 ± 0.63 46.0 ± 0.1  46.6 ± 0.33 WBC (10³/mm³) 5.48 ± 0.41  5.75 ± 1.15  5.86 ± 1.12 platelet (10³/mm³) 685 ± 12  706 ± 28 685 ± 22 MPV (fL) 6.93 ± 0.08  7.2 ± 0.20  7.0 ± 0.09 C-Reactive Protein ND ND ND Antibody to ODN ND ND ND n = 7-9 for each group, ND: non-detectable.

5.7.3 Immune Response

Antibody-antigen reaction was studied by gel immunodiffusion analysis, which revealed no detectable antibody against β₁-AS-ODN. C-reactive protein (CRP), a clinical index for immune reaction, was found negative in all animals.

5.7.4 Tissue Histology

Heart, liver, spleen and kidney were subjected to pathological examination after three repeated injections of β₁-AS-ODN for more than 2 months. No immunopathology or organ damage was found.

5.8 Example 8 DNA Sequences of Known β₁-AR Genes

Particularly preferred antisense sequences for the practice of the present invention include those sequences that specifically bind at or near the region of the AUG translation initiation codon of the mRNA encoding a mammalian β₁-AR polypeptide. Thus, those complementary sequences that are centralized around the region of the mRNA corresponding immediately 5′ of the AUG translation initiation codon are particularly preferred. Such sequences may be identified using a secondary structure analysis (e.g., the OLIGO computer program) to ensure that the oligonucleotides do not fold or self-anneal. The BLAST computer algorithm program may also be used to check the specificity of the chosen sequence. The ordinarily skilled artisan, having the benefits of the teachings of the present invention, can select other suitable antisense sequences using the OLIGO and BLAST programs in combination with the teachings of the subject invention to prepare both oligonucleotides that comprises at least 9 to about 35 contiguous nucleotides that are complementary to a portion of the mRNA encoding the open reading frames designated in each of the genes disclosed in SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, or SEQ ID NO:194.

For the preparation of full-length or substantially full-length antisense polynucleotides, the inventors contemplate the use of sequences corresponding essentially to the entire β₁-AR-specific mRNA sequence produced from one or more of the following β₁-AR genes. For example, the open reading frame of the human β₁-AR gene extends from nucleotide 87 to nucleotide 1520 of SEQ ID NO:187. Therefore, a full-length antisense polynucleotide would consist of an approximately 1.43-knt (kilonucleotide) sequence that is complementary to the region of SEQ ID NO:187 from about position 87 to about position 1520. In order for the complementary sequence to specifically bind to the native β₁-AR-specific mRNA sequence, the polynucleotide need not be exactly the same size, or exactly the same sequence of the native mRNA. In fact, substantially full-length sequences or slightly larger-than full-length are contemplated by the inventors to be as useful in the inhibition of β₁-AR-specific mRNA translation in a host cell as full-length antisense polynucleotides. For example, the size of the complementary polynucleotide may vary (e.g., from about 95% to about 105% of the size of the ORF region of the particular β₁-AR-specific sequence. Thus, for a 1.43-knt full-length mRNA, a sequence of from about 1.36-knt to about 1.43-knt would be considered “substantially full-length” while a sequence of from about 1.44-knt up to and including about 1.50-knt would be considered “slightly larger than full-length,” and complementary polynucleotides may be prepared in either of these size ranges that are sufficiently able to specifically bind to native β₁-AR-specific mRNA sequences in a host cell and inhibit the ability of the host cell's protein synthesis machinery to translate the native β₁-AR-specific mRNA sequence into functional β₁-AR polypeptide.

Such sequences, therefore, would be ideally suited for the preparation of the genetic constructs of the present invention, particularly in their exploitation as gene. therapy vectors for the treatment of the cardiac diseases and disorders of the circulatory system described herein. In the preparation of such gene therapy vectors, one would preferably prepare a genetic construct in which a polynucleotide complementary to the mRNA was placed under the control of a suitable promoter, and then introduce the recombinant vector comprising the genetic construct into the host cells of the affected mammal. Expression of the encoded antisense polynucleotide, therefore, would result in the synthesis of a full-length, substantially full-length, or slightly larger-than full-length polynucleotide that is complementary to the native β₁-AR-specific mRNA produced by the cell. This complementary sequence could then specifically bind to the target mRNA and thereby reduce the availability of native β₁-AR-specific mRNA for translation into mature polypeptide by the cellular protein synthesis machinery.

For the human β₁-AR gene shown in SEQ ID NO:187, exemplary antisense polynucleotide sequences include those that are of from about 1.36-knt to about 1.50-knt in length and that comprise a substantially complementary contiguous sequence selected from about nucleotide 60 to about nucleotide 1600 of SEQ ID NO:187. The selection of substantially complementary, substantially full-length antisense polynucleotides capable of inhibiting the translation of native human β₁-AR mRNA into polypeptide is within the purview of the skilled artisan having the benefit of the present teaching and the selection methods, computer-based complementarity-defining search algorithms, and the assays for β₁-AR mRNA and β₁-AR polypeptides described herein.

In each of the following sequences, the AUG initiation codon is indicated as the first triplet in bold of the corresponding open reading frame.

5.8.1 Human β₁-AR

The polynucleotide sequence encoding human β₁-AR polypeptide is disclosed in Frielle et al. (1987). The GenBank™ Accession No. for the Homo sapiens β ₁-AR gene is NM000684. The Open Reading Frame extends from nucleotide 87 to nucleotide 1520 (shown in bold) (SEQ ID NO:187)

   1 TGCTACCCGCGCCCGGGCTTCTGGGGTGTTCCCCAACCACGGCCCAGCCCTGCCACACCC   61 CCCGCCCCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGGGCGCCTCCG  121 AGCCCGGTAACCTGTCGTCGGCCGCACCGCTCCCCGACGGCGCGGCCACCGCGGCGCGGC  181 TGCTGGTGCCCGCGTCGCCGCCCGCCTCGTTGCTGCCTCCCGCCAGCGAAAGCCCCGAGC  241 GCGTGTCTCAGCAGTGGACAGCGGGCATGGGTCTGCTGATGGCGCTCATCGTGCTGCTCA  301 TCGTGGCGGGCAATGTGCTGGTGATCGTGGCCATCGCCAAGACGCCGCGGCTGCAGACGC  361 TCACCAACCTCTTCATCATGTCCCTGGCCAGCGCCGACCTGGTCATGGGGCTGCTGGTGG  421 TGCCGTTCGGGGCCACCATCGTGGTGTGGGGCCGCTGGGAGTACGGCTCCTTCTTCTGCG  481 AGCTGTGGACCTCAGTGGACGTGCTGTGCGTGACGGCCAGCATCGAGACCCTGTGTGTCA  541 TTGCCCTGGACCGCTACCTCGCCATCACCTCGCCCTTCCGCTACCAGAGCCTGCTGACGC  601 GCGCGCGGGCGCGGGGCCTCGTGTGCACCGTGTGGGCCATCTCGGCCCTGGTGTCCTTCC  661 TGCCCATCCTCATGCACTGGTGGCGGGCGGAGAGCGACGAGGCGCGCCGCTGCTACAACG  721 ACCCCAAGTGCTGCGACTTCGTCACCAACCGGGCCTACGCCATCGCCTCGTCCGTAGTCT  781 CCTTCTACGTGCCCCTGTGCATCATGGCCTTCGTGTACCTGCGGGTGTTCCGCGAGGCCC  841 AGAAGCAGGTGAAGAAGATCGACAGCTGCGAGCGCCGTTTCCTCGGCGGCCCAGCGCGGC  901 CGCCCTCGCCCTCGCCCTCGCCCGTCCCCGCGCCCGCGCCGCCGCCCGGACCCCCGCGCC  961 CCGCCGCCGCCGCCGCCACCGCCCCGCTGGCCAACGGGCGTGCGGGTAAGCGGCGGCCCT 1021 CGCGCCTCGTGGCCCTACGCGAGCAGAAGGCGCTCAAGACGCTGGGCATCATCATGGGCG 1081 TCTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGGTGAAGGCCTTCCACCGCG 1141 AGCTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGGCCT 1201 TCAACCCCATCATCTACTGCCGCAGCCCCGACTTCCGCAAGGCCTTCCAGGGACTGCTCT 1261 GCTGCGCGCGCAGGGCTGCCCGCCGGCGCCACGCGACCCACGGACACCGGCCGCGCGCCT 1321 CGGGCTGTCTGGCCCGGCCCGGACCCCCGCCATCGCCCGGGGCCGCCTCGGACGACGACG 1381 ACGACGATGTCGTCGGGGCCACGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGCTGCA 1441 ACGGCGGGGCGGCGGCGGACAGCGACTCGAGCCTGGACGAGCCGTGCCGCCCCGGCTTCG 1501 CCTCGGAATCCAAGGTGTAGGGCCCGGCGCGGGGCGCGGACTCCGGGCACGGCTTCCCAG 1561 GGGAACGAGGAGATCTGTGTTTACTTAAGACCGATAGCAGGTGAACTCGAAGCCCACAAT 1621 CCTCGTCTGAATCATCCGAGGCAAAGAGAAAAGCCACGGACCGTTGCACAAAAAGGAAAG 1681 TTTGGGAAGGGATGGGAGAGTGGCTTGCTGATGTTCCTTGTTG

5.8.2 Canine β₁-AR

The GenBank™ Accession No. for the Canis familiaris β ₁-AR gene is U73207. The Open Reading Frame extends from nucleotide 330 to nucleotide 1751 (shown in bold) (SEQ ID NO:188)

   1 CCGGGTGCCGGGCCCGCGGGCTCGGCGCGCTCAGAAACATGCTGAGGTCCCGGCGGCTGT   61 TGCAGCAGCGGCAGCGGCTCCAGCAGCGGCTCCAGCAGCGGCTCCGGCAGCGGCTCCAGC  121 GGCGGCAACTCCGGCGGCAGCAGCAGCGGCGGCGGCGGCGGCGGCGGCGCACGGCTCCCG  181 GCGACCTCCGCGCCCCACGTCCCCCGGCGTGGTCCCCGGCCACGGCCCCAGCCCGCCACA  241 GCGACCTCCGCGCCCCACGTCCCCCGGCGTGGTCCCCGGCCACGGCCCCAGCCCGCCACA  301 CCCCCCGCCCCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGCGCTGGCCCTGGGCGCCT  361 CGGAGCCCTGCAACCTGTCGTCGGCCGCGCCGCTCCCCGACGGCGCGGCCACGGCGGCGA  421 GGCTGCTGGTGCCCGCGTCGCCGTCCGCCTCGCCGCTGGCCCCGACCAGCGAGGGCCCCG  481 CGCCGCTGTCGCAGCAGTGGACGGCGGGCATCGGGCTGCTGATGGCGCTCATCGTGCTGC  541 TCATCGTGGCGGGCAACGTGCTGGTGATCGCGGCCATCGCCAAGACGAAGCGGCTGCAGA  601 CGCTCACCAACCTGTTCATCATGTCCCTGGCCAGCGCCGACCTGGTCATGGGGCTGCTGG  661 TGGTGCCCTTCGGGGCCACGATCGTCATGCGGGGCCGCTGGGAGTACGGCTCCTTCCTGT  721 GCGAGCTCTGGACCTCGGTGGACGTGCTGTGCGTGACGGCCAGCATCGAGACCCTGTGTG  781 TCATCGCGCTGGACCGCTACCTGGCCATCACCGCGCCCTTCCGCTACCAGAGCCTGCTGA  841 CGCGCGCGCGCGCGCGGGCCCTCGTGTGCACCGTGTGGGCCATCTCGGCGCTCGTGTCCT  901 TCCTGCCCATCCTCATGCACTGGTGGCGGGCCGGGGGCGACGAGGCGCGCCGCTGCTACA  961 ACGACCCCAAGTGCTGCGACTTCGTCACCAACCGGGCCTACGCCATCGCCTCGTCCGTCG 1021 TCTCCTTCTACGTGCCCCTGTGCATCATGGCCTTCGTGTACCTGCGGGTGTTCCGCGAGG 1081 CGCAGAAGCAGGTGAAGAAGATCGACAGCTGCGAGCGCCGCTTCCTGGGCGGCCCCGCGC 1141 GGCCCCCCGCGCCCCCGCCCGCGCCCGCGCCCGCGCCCCCGCCCGCGCCCGGCTCCCCGC 1201 GCCCCGCCGCGGCCGCCCCGCTGGCCAACGGGCGCGTCGGCAGGCGGCGGCCCTCGCGCC 1261 TCGTGGCGCTGCGCGAGCAGAAGGCGCTCAAGACGCTGGGCATCATCATGGGCGTGTTCA 1321 CGCTGTGCTGGCTGCCCTTCTTCCTGGCCAACGTGGTCAAGGCCTTCCACCGCGACCTGG 1381 TGCCCGACCGCCTCTTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGGCCTTCAACC 1441 CCATCATCTACTGCCGCAGCCCCGACTTCCGCAGGGCCTTCCAGCGCCTGCTGTGCTGCG 1501 CGCGCCGCGCCGCCCGCGGGAGCCACGGGGCCGCCGGGGACCCTCCGCGGGCCCGGCCGC 1561 CGCCGTCCCCCGGGGCCGCCTCGGACGACGACGACGACGACGAGGACGACGCCGGGGCCG 1621 GGGCCGGGGCCGCGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGCTGCAACGGCGGGG 1681 CGGCGGCCGACAGCGACTCCAGCCTGGACGGCGCGGGCAGCCCCGCGGGCGCCTCGGAGT 1741 CCCGGGTGTAGGCGCGCGGCCTCCCGGGGGGCGCATCGGTGTTTACTCCAGACCCAGAGC 1801 AGGTGAACCCCGGGCCCGCGCACCCCCNTCGCATTCATCGAGGCA

5.8.3 Sheep β₁-AR

The GenBank™ Accession No. for the Ovis aries β ₁-AR gene is AF072433/S78499. The mRNA extends from nucleotide 1629 to nucleotide 4749 (underlined). The Open Reading Frame extends from nucleotide 2289 to 3692 (shown in bold) (SEQ ID NO:189)

   1  TCCAGCCCCCTCTTTCTAGCCCTCTCCTTCCCTCATTTCCCCTTCTCAGGCTCCCCAACT   61  GGCAGAACTAAGCTGACAATCCTAAGCCAGGGATGCAGAAACAAGTAATTCACCCACATC  121  CACCCACTGATCATCAAGTTTGGGCCTAAAGCAAATTTACATGTTTGGATAAAGAAAAGT  181  TGGGCTTCCCTAGTAGCTGAGACCCATCTTCAGTCCTTGGATGGGGGAAGATCCCCTAGA  241  GAAGGAGATGGCAACCCACTCTAGTATTCTTGCCTGGAAAATCCCATAGGCAGCGGAGCC  301  TGGTGGCTACAGCCCATGGGGTTGCAAGAGTCAGACACAACTTAGCTACTAAAACCACCA  361  CCCATGGCTTATGAATACACATTGCTGTTAGCTCTCGACTTAGGGAGCTCTCTCCAAGGT  421  AAGAATATGAGTTTGTTCCTTTCAGAAACTATTCTTTTTATTCCAATGCTAGAAGGATGT  481  GTGAGCATTATGTAACATTTTCATGCACCCTTAAGTGGGTAATTAGAAGCTCTTTATTTC  541  TCAGGATTCAATTAAAAGCTTTTTATTTTCAAGGCTGAGTTGAGGACCAGTACTGTGGTG  601  GAATTAGACAAGGGGCTTGCACACCTTTGGCTACATTGTGTGTTGATGGGCCACCTTCCT  661  GTAGGTACCTCCCCACATATAGTCACACCACTGCAGAGCTAACGACTCACTAATTTTAAA  721  CCCATTCAGTTGCCAACCCAACAGCCTTTGATATAACTTTACATGCTATTGGATTTTAAT  781  CTTTTTGAGTATTTATATATGTTTTCTTCTCTCATCCCTCCAAAATTAATCCTAGAGTTT  841  TGAGAATCTGGGAACTTGGGCAAAGGAGAAGGCAACGCAGCAGACCAAGAAATTTGAAAT  901  CTCAGTTCACTACTGTGTCACCCAAAGTCAATGTACCTTTTTTGTTTGGACCGGCCCAGC  961  TCAAGTCATACAATCACGTGAGTAACAGACCACAAAATCCAGGTGTTATTACTGAACATG 1021  ACAAGTCTGAAAAGTAATTACACGTGTTCTAGCTTCCGTGGCGGTGTCATTTACTCTAAC 1081  ATGCCTGTCCTTAAGCCTCTCTCTCTCTTTACATTACCGGCACACACCGGTGCACCATAC 1141  TCACACATCCATCAGCTGGGACCTGGGAGTGTGTATTATTCCAACTGGTCCTCAGCATTA 1201  GCTGTCAGATGTCACAACCCCCYGCCGTTTTCTGCATCTGCTGCCCCGGGAAGCGAGAAG 1261  AAGCTTGCAAGAATAGCTCCCGGGAACGTTCCTGAAAGATTGGCGCTCTGCTTTAGCAAG 1321  GCGCTCGCTGGAAAGTTTCTTCTAACCGCTCACACCCGCCTCCGATCCGATCCCCGAGCT 1381  GGCAGGACGCGAGCTGGCTGGGACTCCTCTTGACAGAGGAAGGGCTTTACACACCACCCT 1441  CCTAGGCTGCCCAATACAAGAAACAGTCTTGCAGCCAGACTCCTCCACACCCAGCGAACA 1501  GACCGTCCAAGGCGCTCCGGTGTTTCGAGAACACCGAAGTCCCCTCCCTGCTAAAGGGCG 1561  CGTGAGCTCTGCTCTGCAGGAAACCTGGGCACTGGAGGTAGATGGGATGGGTGGCGGCGG 1621  GTAGAGCCGGGGCGCAGCGGAAAGCAAACGCCGGAGGCAAACGGGGCGCAGGAGAGGGGA 1681 GATTGGGTGCCGCCGTAGGGGCCAGGGTGAAAGCCGGGCGCGGACGGGAACCGAGGGGAA 1741 CTGGGCACTGGAGCCAAGCGGGCTCTGGAAGGGACGCGCGGGCAGGAACCCGCGAGCGCT 1801 GGGGAGGGGCTTGCTTGGCGATCTGCCCCGGACTCCCTAGAGCCGCAGAACCGCCGGTGG 1861 AGGCGGGGTGCTAGGAGTTGGCGGGGCCGGGTGGGGGTGGGGGGGAACCAGAGAGGGGCG 1921 TGCCTTCGCCAGGATTGGCTGCAGGAGCCTGACGCGAGNNNCCGGGGGTTGGCTCGGGGG 1981 AGTGGGAGCCGGGTGGGGTGGGTGCTGGGTGCCGGGGCTGCGGGCTCCGCGAGCTCAGAA 2041 ACATGCTGAGGTCCCGGCAGCTGTTCCAGCAGCGACACCACTCCAGCAGCAGCCGCGGCG 2101 GCTGCGGCGGCGACAGGCACCGGCTCCGGCGGGGAAGGCGCCCGGCGCCATGCCTCCGGC 2161 CCCGCGCCGCGCTGCGCTGACCTGGCCGCGACCTCCCTCCGCGCGCCCCGCCGTTCGGGC 2221 CTCTGGGGGGTTCCCCAACCGCGGCCCAACTCCGCCACACCCCTCTCCCCCGGCCTCCGC 2281 AGCTCGGCATGGGCGCGGGGGCGCTCGCCCTGGGCGCCTCCGAGCCCTGCAACCTGTCAT 2341 TCGCCGCGCCGGTCCCCGACGGCGCGGCCACGGCGGCGCGGCTGCTAGTTCCCNCGTCGC 2401 CGCTCCGCCTCGCTGCTGACCTCGGCCAGCGAGGGACCCCGCTGCTGTCGCAGCAGTGGA 2461 CGGTCGGCATGGGCCTGCTGATGGCATTCATTGTGCTGCTCATCGTGGCGGGCAACGTGC 2521 TGGTGATCGTGGCCATCGCCAAGACTCCGCGGCTGCAGACGCTCACCAACCTCTTCATCA 2581 TGTCGCTGGCCAGCGCAGACCTGGTCATGGGTCTGCTGGTAGTGCCGTTTGGAGCCACAA 2641 TCGTGGTGTGGGGCCGCTGGGAGTATGGCTCCTTCTTCTGCGAGCTCTGGACCTCGGTGG 2701 ACGTGCTGTGCGTGACGGCCAGCATCGAGACCCTGTGTGTCATCGCCCTGGACCGCTACC 2761 TCGCCATCACGTCGCCCTTCCGCTACCAGAGCCTGCTGACCCGCGCGCGAGCGCGGGCCC 2821 TCGTGTGCACCGTGTGGGCCATCTCGGCGCTGGTGTCCTTCCTGCCCATCTTCATGCAGT 2881 GGTGGGGGGACAAGGACGCCAAGGCGAGCCGGTGCTACAACGACCCCGAGTGCTGCGACT 2941 TCATCATCAACGAGGGCTACGCGATCACCTCTTCCGTCGTCTCCTTCTACGTGCCCCTGT 3001 GCATCATGGCCTTCGTGTACCTGCGGGTGTTCCGCGAGGCCCAGAAGCAGGTGAAGAAGA 3061 TCGACAGCTGCGAGCGCCGCTTCCTCAGCGGCCCCGCGCGGCTGCCCTCGCCCGCGCTCT 3121 CGCCCGGGGCGCCGCTCCCTGCCGCCGCGGTGGCCAACGGGCGCGCCAACAAGCGGCGGC 3181 CCTCGCGCCTCGTGGCCCTGCGCGAGCAAAAGGCCCTCAAGACGCTGGGCATCATCATGG 3241 GCGTGTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGGTGAAGGCCTTCCACC 3301 GCGACCTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGG 3361 CCTTCAACCCCATCATCTACTGCCGCAGCCCCGACTTCCGCAAGGCCTTCCAGCGCCTGC 3421 TCTGCTGCGCGCGCCGGGCCGCCTGCGGGAGCCACGGGGCCGCCGGGGACCCGCCGCGCG 3481 CCGCGGGCTGCCTGGCGGTGGCCCGGCCGTCGCCGTCTCCCGGGGCCGCCTCGGACGACG 3541 ACGACGACGACGACGAAGACGACGTCGGGGCCGCGCCGCCCGTGCGCCTGCTGCAGCCCT 3601 GGGCTGGCTACAACGGCGGGGCGGCGGCGAACAGCGACTCGAGCCCGGACGAGCCAAGCC 3661 GCCCGGGCTGCGGCTCGGAATCCAAGGTGTAGGGACGGGCGCCCCTCCCCGCCTTCCCCG 3721 GCTTCCCCAGTCCGGGAGCGGGCTGTGCGCTCCAGGAGCAAGAGAACCCGGGCGMCCCTG 3781 AACCGCTTCCCCGGGAAAGAGGTCTGTGTTTACTCGAGACCGTAAAGCAGGTGAACTCGA 3841 AGCCTGCGAACCTCGTCTGCATCATCCAAGGGCAAATAGGAAAGCCACGGACCGTCGCAC 3901 AGAAAGGAAAGTTTGGGGAGAGGTGGGAGAAGTTTGGGGAGGGGTGGAAGAGTGGCTTAC 3961 TGATTGTTCTTGGGGTTCTTTTTCCTGTTTCTGGTCCAGCCTTCTGTGTGTGCGTGTGAT 4021 GCATCTTTAGAGTCCCTCCTCCCCCCGCCCCCCCGCGACGTGGCTTTTAACACTTTCTGC 4081 GATGACTGGGAAGGGAAGGGGGAAGCGTTAGGAGGTAAAAGTCTCTCGACTTAGTTTCCA 4141 TCCCATTCCTGGGAACAGAAGCGGTCAGCCAGAGAGAGAGGAGAGAGAATGACACTTTAT 4201 CAGGACGTTGTTTCCTTTTGCTTTTCAGAGAAATACCATTTTAATTTCTGAGGAATTATT 4261 TCTCCTGTTCTGAAAGCCGAGGGCAAGGATGGATGCAAAAATCGCGTTTCAGGAAGTTTT 4321 ATGCTCTTCTTGGAACAAGCCTCACCTTGCTTCCCTTTCGGAGGGCAAGCGGGCTGTCCC 4381 TGAACGCCTCCTCGGTGGTCAGGCTGAGGGGTTCCTACCTCACTCACACGGTGCACATTG 4441 CACGGCCAGATAGAGAGACTTGTTTATATTAAACAGCTTATTTATGTATCAATACTAGTT 4501 GGCCGGACCAGGCGCTGAGCCTTCGTGACATGTGACTCTGTCCATTGAAGACAGGACAGA 4561 AAAAGGAAAAAGAAAAAAGGAAAACAATTCAGATTACTGCACATGTGGTATAGACAAAAA 4621 ATCAAAACAAAAAAGCCGTGATTCAAAGTGGCATTTTTTTTGCACAGTATTAGGAACTGT 4681 AAAGTCCACAGAAAATGTTATTTGCACAAAAAGAAATGAAATATTTTTTAATGGGAGTGG 4741 GGTGGGGCA

5.8.4 Porcine β₁-AR

The GenBank™ Accession No. for the Sus scroffa β ₁-AR gene is AF042454. The Open Reading Frame extends from nucleotide 456 to nucleotide 1862 (shown in bold) (SEQ ID NO:190)

   1 GACTCCCCAGAGCTGCTGAACCGCCGGGGGGAGGGGGTGGGGGAGTTGGCGGGGGGG   61 GGGGGGGAGAACCAGATCGGGGCGTGCTTCCTCGGATTGCTGCAATAGCTGACGCGA  121 GGCCCCGGGGGTTGGCTCCAGGGAGTGGGATGGGAGCGGGTGGGGTGGTGCTGGGTG  181 CCGGGGCTGCGGGTTCCGGCGCTCAGAAACTGCTGAGGTCGCAGCTGTCCAGCAGCG  241 ACACCGCTCCAGCAGCAGCGGCGGCGGCGGGGCGGCGACGCGCACAGCCTGGCGGGG  301 AAGGCGCCCTGCGCCCATGCCTCCGGCCCCCGCCGCGGCGCCCTGACCGGCCGCGAC  361 CTCCCTCGTCGCGCCCCGCCGCCCGGGCCTTGGGGGGGTCCCGACCGCGCCCAACTC  421 CGCCACACCCCCCGCCCCCGCCTCCGCAGCCGGTATGGGGCGGGGGCGTCGCCCTGG  481 GTGCCTCCGAGCCCTGCAACCTGTCATCGGCGCGCCGCTCCCGACGGCCGGCCACCG  541 CGGCGCGGCTGCTGGTGCCTGCGTCCCCTCCGCCTCGCTCTGACCCCACCAGCGAGG  601 GATCCGTGCAGCTGTCGCAGCAGTGGACGGTGGCATGGGCTCCTGATGCGCTCATCG  661 TGCAGACGCTCACCAACCTCTTCATCATGTCCTGGCCAGGCCGACCTGTCATGGGGC  721 TGCAGACGCTCACCAACCTCTTCATCATGTCCTGGCCAGGCCGACCTGTCATGGGGC  781 TGCTGGTGGTGCCATTCGGGGCCACCATCGGGTGTGGGGCGCTGGGAGACGGCTCCT  841 TCTTCTGCGAGCTCTGGACGTCGGTGGACGACTGTGCGTACGGCCAGTTCGAGACCC  901 TGTGTGTCATCGCCCTGGACCGCTACCTCGCATCACGTCCCCTTTCGCACCAGAGCC  961 TGCTGACCCGCGCGGCACGGGCCCTCGTGTCACCGTGTGGCCATCTCGCCCTGGTGT 1021 CCTTCCTGCCCATCCTCATGCACTGGTGGCGGACAAGGGGCCGAGGCAGCCGCTGCT 1081 ACAACGACCCCAAGTGCTGCGATTTCGTCACAACAGGGCTACGCCATCCCTCGTCCG 1141 TGGTCTCCTTCTACGTGCCCTTGTGCATCAGGCCTTCGTTACCTGCGGTGTTCCGCG 1201 AGGCCCAGAAGCAGGTGAAGAAGATCGACACTGCGAGCGCGCTTTCTCGCAGCCCCG 1261 CGCGGCCGCCCTCGCCCGCGCCCTCGCCCGGTCCCCGCTCCTGCCGCCCTGCCGCAG 1321 CCCCGGTAGCCAACGGGCGCACCAGCAAGAGCGGCCCTCCGCCTCGTGCCCTGCGAG 1381 AGCAGAAGGCGCTCAAGACGCTGGGCATCACATGGGCGTTTCACGCTCGCTGGCTAC 1441 TCGTCTTCTTCAACTGGCTGGGCTACGCCACTCGGCCTTAACCCCATCTCTACTGCC 1501 TCGTCTTCTTCAACTGGCTGGGCTACGCCACTCGGCCTTAACCCCATCTCTACTGCC 1561 GCAGCCCTGACTTCCGCAAGGCCTTCCAGCCCTGCTCTGTGCGCGCGCGGGTCGCCC 1621 GCGGGAGCTGCGCGGCCGCCGGGGATGGGCGCGCGCCTCGGCTGCCTGCGGTGGCCC 1681 GGCCGCCGCCGTCGCCCGGGGCCGCCTCGGCGACGACGAGACGAAGAAACGTCGGGG 1741 CCGCGCCGCCGGCGCCCCTGCTGGAGCCCTGGCCGGATAAACGGCGGGCGGCACGTG 1801 ACAGCGACTCGAGCCTGGACGAGCGGACGCCGGGGGCCGGCCTCGGAACCAAGGTGT 1861 AGGGCCCAGCGCTCCCTCCCCACCTCCCAGGGGGATGCAGCTCTGCGCGAGAGAAGA 1921 GAACCCGGCGCCCCGAAAGGCTTCCCGGGGATGAGGAGACTGTGTTTATCGAGACCG 1981 AAAGCAGGTGAACTCGCAAACCTCGTCTGCTCATCTAAGCAAACAGAAAGCCGGACC 2041 GCTGCACAGAAAGGAAAGTTTGGGGAGGGTGGAGAGTGGTTGCTCATTTTGTTGAGT 2101 TCTTTTCTCTGTTTGTGGTCCGTCCTCCTTTGTGTGTGCTGTGATGCACTTTAGATT 2161 TTTATTCCCCCAGGTGGTTTTTAACACTTTTGCGAAGACAGGGAAGGGTGGGAGAAG 2221 CAGGAGAGTTTTTAAAAAGGTGTCTCAACTGGCTTCCATCCGTTCCCGGGACGGAGC 2281 AGTC

5.8.5 Rat β₁-AR

The rat β₁-AR polypeptide and polynucleotide sequences are disclosed in Machida et al. (1990). The GenBank™ Accession No. for the Rattus norvegicus β ₁-AR gene is D00634. The Open Reading Frame extends from nucleotide 1257 to nucleotide 2657 (shown in bold) (SEQ ID NO:191)

   1 GGTACCAGAGTACAAACGTCGGTGTTAAGGTGTTGGTACTGGAGTCAAAAGTCTGAAG   61 CGTCATTCTCTGAGTTCTTTTACGCGGGGTGGGGGTTGGGGGTGGGGCGTGTCATTTA  121 CTCCGGACTTAATGTTGCCCAAACTTCTCCTTTATACTCAGAACTCATCCATCCTTTT  181 CCTTAGCTGGGATTGGGAACGTGTGTGTCCAACTGGTCCTTGGCGTTGGAGTTACAGC  241 CACAAACCTTCCCTTCCTTCCCCATCCACTGCACTTGAGGGAGAGAGAGACAGCTCTG  301 CGGAAGAACAGCGCCCAGAGGCCTTTCTGACAGATGGCACTCTGCTCCCCAATCCGCC  361 TGGCTGCAAAGTTTCCTCTATAACTAACACCCACTTCCTATTCCCCAAGTGTCATTCT  421 GGCCGGGTCCCCTCGCGATCGGGAAGAGCCAGGCTGCCTGATGGCAGAACAGTCTCAC  481 AGCTAAATTCTCTAATCCCACAGATAGGCTGTCCAAGGCATCTCCGGAAGCCAGCAAG  541 TTTCCCTTCTAGCTACCAGGACCATGCACTAGAGGTAGAAAGGCGAGGGGCTACGGGG  601 CGCCGAAGGGAAACCGGCTCTGCGGGAGGGCAAAATGAGAGCTGGGTGCAGGGCAAGC  661 GGACACCACTTGGGCGGTGGGGTGGGCACAGGAGGCCGGCGGGGCACCCAGAGGGCGC  721 AGCAGACCAGACTCTGGAAGAGCCTGAGCAGGAAAGGGCGCGCTCGCTGAGCCCGTGG  781 GTGCCGGCTGCGTAGTCCACCGCAATCTTTGGAGCCTTTCAATCGCGGTGGAGGGGGT  841 GCTGGGTGTTGGGGGTGGGGCACCATTGTTCGGGGGCGTGCCTTGGACGCGATTGGCT  901 GCGGGAGCCTGACGCGCGGCCCGGGGGCTGGCTGGGGGGTAGGGAGCGAGTGGGGGGG  961 AGGTGCTGGGTGTTGGAGCCCCGGCCCCGCGCGCTCAGAAACATGCTGAGTCCCGGCA 1021 ACTCTTCCAGCAGCGACCCGTCCAGCAGCAGCGGCGGCGGCGGCGGCGGGACACGGCT 1081 TGGCTACGGAGGAGAAGGCGCCGGCGTCCATGCCTCCGGCCCCAAGCCGGGCTGCCCT 1141 GACCTGGCCGCGACCTCCCCCGTCCCCGCGCGCGCCCCCAGCCCCGGCTCTGGGGTGC 1201 TTCCCAGGCGCGGCCCAGTCCGCCACACCCCCCGCGCCCGGCCTCCGAATCGGCATGG 1261 GCGCGGGGGCGCTCGCCCTGGCGCCTCCGAACCCTGCAACCTGTCGTCGCCGCGCCGC 1321 TGCCCGACGGCGCGGCCACCCGGCACGACTGCTGGTGCTCGCGTCGCCTCCGCCTCGC 1381 TGCTGCCTCCAGCCAGCGAGGCTCAGCGCCGCTGTCGCAGCAGTGGACCCGGGTATGG 1441 GCCTACTCCTGGCGCTCATCTGCTGCTCATCGTAGTGGGCAACGTGTTGTGATCGTGG 1501 CCATCGCCAAGACCCCGCGGTGCAGACGCTCACCAACCTCTTCATCATGCCCTGGCCA 1561 GCGCCGATCTGGTCATGGGATGCTGGTGGTGCCTTTCGGGGCCACCATTTGGTGTGGG 1621 GCCGCTGGGAGTACGGCTCCTCTTCTGTGAGCTCTGGACTTCGGTAGACTGCTATGTG 1681 TGACGGCCAGCATCGAGACCTGTGTGTCATCGCCCTGGACCGCTACCTCCCATCACGT 1741 CGCCCTTTCGCTACCAGAGCTGCTGACGCGCGCGCGAGCGCGGGCCCTCTGTGCACAG 1801 TGTGGGCCATCTCCGCGCTGTGTCCTTCCTGCCCATCCTCATGCACTGGGGCGGGCCG 1861 AGAGCGACGAAGCGCGCCGCGCTACAACGACCCCAAGTGCTGCGATTTCTCACCAACC 1921 GGGCCTACGCCATCGCCTCGCCGTCGTCTCCTTCTACGTGCCCCTGTGCTCATGGCCT 1981 TCGTGTACCTCCGGGTGTTCGCGAGGCCCAGAAACAGGTGAAGAAGATCACAGCTGCG 2041 AGCGCCGCTTCCTCAGCGGCCGCCCCGGCCGCCCTCGCCCGCGCCCTCGCATCACCAG 2101 GGCCACCGCGCCCCGCAGACCGCTGGCCAACGGGCGCTCCAGCAAGCGGGGCCGTCGC 2161 GCCTCGTGGCTCTGCGAGAGAGAAGGCGCTCAAGACACTGGGCATCATCTGGGTGTGT 2221 TCACGCTCTGCTGGCTGCCCTCTTCCTGGCCAACGTGGTGAAAGCTTTCACCGCGACC 2281 TGGTGCCGGATCGCCTCTTCTCTTCTTCAACTGGCTGGGCTACGCCAACCGGCCTTCA 2341 ACCCCATCATCTACTGCCGCGCCCCGACTTCCGCAAGGCTTTCCAGCGCTGCTTTGCT 2401 GCGCGCGCCGGGCCGCCTGCGACGCCGCGCAGCCCACGGGGACCGGCCGGCGCCTCGG 2461 GCTGCCTGGCGAGAGCTGGGCGCCGCCGTCCCCCGGGGCTCCTTCGGACACGACGACG 2521 ATGACGCCGGGGCCACCCCACCGCGCGCCTGTTGGAGCCCTGGGCCGGCGCAACGGCG 2581 GGACGACCACTGTGGACAGCATTCGAGCCTGGACGAGCCGGGACGCCAGGCTTCTCCT 2641 CCGAGTCCAAGGTGTAGAGGCCAGGCTCTCCGGGCGCACGGACGCCGCTCCCATAGTC 2701 CCGGGCTGGACACGGGCTCTCATCCCTAGAGGAAGAGAGAAAGAATGGGCCCTGAGCC 2761 GCTCCCCAGGGGAGAGAGGAATTTCTGTTTACTCAAGACCGAAAGCAGGGAATGCGAA 2821 GCCCACAGATCTTTTGAATCTCCGAGACGTACAGAAAAGCCCCGGACCGGCGTTGCGC 2881 AAAAAGGAAAGTTTGGGAGTGTTGGGAGAGTGTGGCTTAGTGTGGCTTATGGCTTGTC 2941 TTGAGTTCCTTTCTCTCTGAGTTCGGCCTTTCGTGTGTTTAATGCACCTTAGGCACCC 3001 CCCCCCCGTGGGTTTTGACATTTCTGCAAGGACCCGAGTGGAAACTAGGGGGGAGGGG 3061 AAGGGGGAGGGTGGAGTCTACACCTGCCTTCTACTTCACACCTAGGAACTAAGTGTTC 3121 AGCTCTGGTTTGGGGTGGGCCAGGAGCGGTGATAATTAGCCAGGAAGTGTCCTTTTGC 3181 TTTCTAAAGAAATTGCATATAATTCCGGAGTATTGGTGTCTCCTTAAAGAAAGGGGGG 3241 AAAGGTGGCTGAGAAACAGACAATCTGGTTTCGAGAAACTATTTGTGGACACGGTTCA 3301 CCTTGCTTTCTCCTGGAGGGAAACCCTGTCCCTGCGCGCCTCGGTGGTCTGCTGTGGG 3361 TCCTCTACCTCACTCTGTGCTATTGCACAGCAAGATAGAAAGACTTGTTATATTAAAC 3421 AGCTTATTTATGTATCAATATAGTTGGAAGGACCAGGCGCTGAGTCTCTTCTGTGACA 3481 TGTGATTCTGTCAACTAAAGTAGGACAGAACAAAAGGAAACAGTTGGGATATTGCACA 3541 TGTGGCTAAAAACAAAGATGCAAAAAAAAAAGGCAGTGGTTGAAAGGCCTTTTGCGCA 3601 GTGTTAGACATTGTAAAATCATAGAAGTTGTTAACTTGCACAAAAAATTAATATTTTT 3661 AATGGGACGGGGAGGTGGGCGATCT

5.8.5 Rhesus Monkey β₁-AR

The GenBank™ Accession No. for the Macaca mulatta β ₁-AR gene is X75540. The Open Reading Frame extends from nucleotide 1425 to nucleotide 2867 (shown in bold) (SEQ ID NO:192)

   1 CCAAGGAATCTGAAATCGCAGTTCATGACGTCAGCATAAACCGACAGTACCTTTTTGTTT   61 GTATTGACCCAGCTCAAATTATAAAATCACATGAGTAATAAAACACAAAATACAGGTGTT  121 GTCACTGAAAAGCCTAAAATGTAACTAAACGCGTTTTCCCCCCTCGCGGTGGTATCATTT  181 ACTCTGTATCTCAACGTTTCTGTCTCCCTAAGACTCTCCCTTTATATCGTGAGCACACAT  241 TTTTGCATCATGCTCACACATTCATTAGCTAGGACTGAGGAGTGTGTACGATTCCAACGG  301 GCCCTCAGCGTTAGCTGTTAGATGCACAAACCTTCACTTCCTTTCCACATCTACTGCACT  361 TGAGGTTCAACAGAGGATCCTTGCAAGAACAGTGCCCAGAGAGCATTCTTGACAGATGCG  421 CGCTTAGCTCCAGCAACCCGCTCTGCTGGGAAGTTTCTTCTAACCACTAACACCCACCTC  481 CAATCCCCCAAGCTGTCACGACGCAGGCTGGCTGGGTCCCGTATTGACGGGGGAAGGGTT  541 TTACACACATCCTGCTAGGCTGCCCCACATCACAACCAAGCTCGCAGGGCAAACTCCTCC  601 AAGCCTGGCGGACAAGCTGTCCCAGGCGCTCTGGCGCTTCCTGAACACCAAGGTCCCCTC  661 CGCGCTCAAGGGAGCTAGCGCACTGTTACGCAGGGAACCCCGGCACTGCAGGTCGAGGGG  721 ATGCCGAGGGAGCGGGGGCGCAGCAGGCAGCCGACTGCTGTAGGCAAACGGCGCGCAGGA  781 GGCAAACGAGGCGCAGGAGCCGGTGCGAGAGCGAGTGGGCGCTGAGAGAGGGGGCGGGGC  841 CCCAGGGGGGAGGCGAGCGCGGGAGGGGGCACGGGAGAACAGGGACCAGGAACCAGCGGG  901 CGCAGGAAGGGGTGCGTCCGCAAGAACCCGCGGGCGCACGGGAGGCACTAGCTCCACGAT  961 CAGCTCGGGACTCTCAGGAGCCGCTCAATTGCCAACGGGAGGGGGCTGCGGGGAGTTGGA 1021 GGTTGGGGGGGCTGACCAGACGGGGGCGTGCCTTTGCCCGGATTGGCTGCAGGAGGCTGA 1081 CGCGAGGCCCCGGGGGTTGGCTTGGGGAGTGGGAGCGGGGTGGGGTGGGTGCTGGGTGCC 1141 GGAGCTGCGGGCCCGGCGCGCTCAGAAACATGCTGAGGTCCCGGCGGCTCTTCCAGCAGC 1201 GGCAGCGGCTCCAGCAGCAGCGGCGGCGGCGGCGGCGGCGGCAGCGACAGCGCTCGGCTC 1261 CGGCGGGAAAGGCGCCCGGCGCCCATGCCTCCGGCCCCGGGCCGCGGCTGCCCTGACCCG 1321 GCCGCGACCTCCCTCTGCGCACCCCGCCGTCCAGGCTTCTGGGGTGTTCCCCAACCAAGG 1381 CCCAGCCCTGCCACACCCCCCGCCTCCGGCCTCCGCAACTCGGCATGGGCGCGGGGGCGC 1441 TCGTCCTGGGCGCCTCCGAGCCCGGTAACCTGTCGTCGGCCGCACCGCTCCCCGACGGCG 1501 TGGCCACCGCGGCGCGGCTGCTGGTGCCCGCGTCGCCGCCCGCCTCGTTGCTGCCTCCCG 1561 CCAGCGAAGGCCCCGAGCCGCTGTCGCAGCAGTGGACGGCGGGCATGGGTCTGCTGATGG 1621 CGCTCATCGTGCTGCTCATCGTGGCGGGCAACGTGCTGGTGATCGTGGCCATCGCCAAGA 1681 CGCCGCGGCTGCAGACGCTCACCAACCTCTTCATCATGTCCCTGGCCAGCGCCGACCTGG 1741 TCATGGGGCTGCTGGTGGTGCCGTTCGGGGCCACCATCGTGGTGTGGGGCCGCTGGGAGT 1801 ACGGCTCCTTCTTCTGCGAGCTGTGGACCTCGGTGGACGTGCTGTGCGTGACGGCCAGCA 1861 TCGAGACCCTGTGTGTCATCGCCCTGGACCGCTACCTCGCCATCACCTCGCCCTTCCGCT 1921 ACCAGAGCCTGCTGACGCGCGCGCGGGCGCGGGGCCTCGTGTGCACCGTGTGGGCCATCT 1981 CAGCCCTGGTGTCCTTCCTGCCCATCCTCATGCATTGGTGGCGGGCGGAGAGCGACGAGG 2041 CGCGCCGCTGCTACAACGACCCCAAGTGCTGCGATTTCGTCACCAACCGGGCCTACGCCA 2101 TCGCCTCGTCCGTGGTCTCCTTCTACGTGCCCCTGTGCATCATGGCCTTCGTGTACCTGC 2161 GGGTGTTCCGCGAGGCCCAGAAGCAGGTGAAGAAGATCGACAGCTGCGAGCGCCGTTTCC 2221 TCGGCGGCCCCGCGCGGCCGCCCTCGCCCTCGCCCTCGCCCTCGCCCTCGCCGGTCCCCG 2281 CGCCGCCGCCCGGACCTCCGCGCCCCGCCGCCGCCGCTGCCACCACCGCCCCGCTGGTCA 2341 ACGGACGTGCGGGTAAGCGGCGGCCCTCGCGCCTCGTGGCCCTGCGCGAGCAGAAGGCGC 2401 TCAAGACGCTGGGCATCATCATGGGCGTGTTCACGCTCTGTTGGCTGCCCTTCTTCCTGG 2461 CCAAACTGGTGAAGGCCTTCCACCGCGAGCTGGTGCCCGACCGCCTCTTCGTCTTCTTCA 2521 ACTGGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCATCTACTGCCGCAGCCCCGACT 2581 TCCGCAACGCCTTCCAGCGACTGCTCTGCTGCGCGCGCAGGGCTGCCCGCCGGCGCCACG 2641 CGGCCCACGGAGACCGGCCGCGCGCCTCGGGCTGTCTGGCCCGGCCCGGACCCCCGCCGT 2701 CGCCCGGGGCCGCCTCGGACGACGACGACGACGATGTCGTCGGGGCCACGCAGCCCGCGC 2761 GCCTGCTGGAGCCCTGGGCCGGCTGCAACGGCGGGGCGGCGGCGGACAGCGACTCGAGCC 2821 TGGACGAGCCGTGCCGCCCCGGATTCGCCTCGGAGTCCAAGGTGTAGGGCCCGGCGCCGG 2881 GCGAGGACGCCGGGCACCCCGGGAGGAGGAGAGCCCGGGCGCCCCGGAACGACTTCCCGG 2941 GGGAACGAGGAGATCTGTGTTTACTCAAGACCGAAAGCAGGTGAACTCGAAGCCCACAAT 3001 CCTCGTCTGAATCATCCGAGGCAAACAGAAAAGCCACGGACCATTGCACAAAAAGGAAAG 3061 TTTGGGGAGGGATGGGAGAGTGGCTTGCTGATGTTCCTTGGTTTTTTTTCTTTCTTTCTT 3121 CCTTTTTTTTTTTTTTTTTCTGTTTGTGGTCCGGCCTTCTTTTGTATGTGCGTGTGATGC 3181 ATCTTTAGATTTTTTTTCCCCCACGTTGGTTTTTGACACTCTCTGCGAGGACCGGAGTGG 3241 AAGTTGGGTGGGTTACGGGAAGGAAGAAGCATTAGGAGGGGATTAAAATCGATCAGCGTG 3301 GCTCCTATCCCTTTCCCAGGAACAGGAGCAGTCTACCAGCCAGAGGGAGGAGAATGACAG 3361 TTTGTCAAGACGTATTTCTTTTGCTTTCCAGATAAATTTCATTTTAATTTCTAAGTAATG 3421 AGTTCTGCTGTCATGAAAGCAAAGAGAAAGGATGGAGGCAAAAAAAAAAAAATTCACGTT 3481 TCAAGAAATGTTAAGCTCTTCTTGGAACAAGCCCCACCTTGCTTTCCTTGTGTAGGGCAA 3541 ACCGGCCGTCCCCCGCGCGCCTGGGTGGTCAGGCTGAGGGATTTCTACCTCACACTGTGC 3601 GTTTGCACAGCAGATAGAACGACTTGTTTATATTAAACAGCTTATTTATGTATCAATATT 3661 AGTTGGAAGGACCAGGCGCAGAGCCTCTCTCTGTGACATGTGACTCTGTCAATTGAAGAC 3721 AGGACATTAGAGAGAGAGAAACAGTTCAGATTACTGCACATGTGGATAAAAACAAAAACA 3781 ACAAAAAAAGGAGTGGTTCAAAATGCCATTTTTGCACAGTGTTAGGAATTACAAAGTCCA 3841 CAGGAGATGTTACTTGCACAAAAAGAAATTAAATATTTTTTAAAGGGGGAGGGGCTGGGC 3901 AGATCTTAAAAACTAAAATAAAATTCAAACTCTACTTCTGTTGTCTAGTATGTTATTGAG 3961 CTAATGATTTATTGGGGAAAATACCTTTTTATACTCCTTTATCATGGTACTGTAACTGTA 4021 TCCATATTATAAATATAATTATCTTAAGGATTTTTATTTTTTTTTTTTTTATGTCCAAGT 4081 GCCCACGTGAATTTGCTGGTGAAAGTTAGCACTTGTGTGTGAACTCTACTTCCTCTTGTG 4141 TGTTTTACCAAGTATTTATACTCTGGTGCAACTAACTACTGTGTGAGGAATTGGTCCATG 4201 TGCAATAAATACCAATGAAGCACAATCAAGATTATGTACTGTGTGTCTGTAAAGGGTCAG 4261 TGATAATGAAAAAGACAGTTTGTTTTGTTCAAAATATAGACTGGATTTCCCATAGAGCTC 4321 TTTTAATAGACTTTCATGACTCAATAACATAGCAAAATGCCTCCAGACCTAAATAAGGTG 4381 TTTACCTACTGAGAGCTGCAG

5.8.6 Mouse β₁-AR

The GenBank™ Accession No. for the Mus musculus β ₁-AR gene is L10084. The Open Reading Frame extends from nucleotide 100 to nucleotide 1500 (shown in bold) (SEQ ID NO:193)

   1 GACCTCCCCGCGCGGGCCCCGCAGCCCCGGCTCCTGGGGTGCTCCCCAGGCGCGGCCCAG   61 CCCCGCCACACCCCCCGCCCCCGGCCTCCGCAGCTCGGCATGGGCGCGGGGGCGCTCGCC  121 CTGGGCGCCTCCGAACCCTGCAACCTGTCGTCCGCCGCGCCGCTGCCCGACGGTGCGGCC  181 ACCGCGGCGCGGCTGCTGGTGCTCGCGTCGCCTCCCGCCTCGCTGCTGCCTCCAGCCAGC  241 GAGGGCTCAGCGCCGCTGTCGCAGCAGTGGACCGCGGGTATGGGCCTACTCGTGGCGCTC  301 ATCGTTCTGCTCATCGTGGTGGGTAACGTGCTGGTGATCGTGGCCATCGCCAAGACCCCG  361 CGGCTGCAGACGCTCACCAACCTCTTCATCATGTCCCTGGCCAGCGCTGATCTGGTCATG  421 GGATTGCTGGTGGTGCCTTTCGGGGCCACCATCGTGGTGTGGGGCCGCTGGGAGTACGGC  481 TCCTTCTTCTGCGAGCTCTGGACTTCGGTAGATGTGCTGTGTGTGACGGCCAGCATTGAG  541 ACCCTGTGTGTCATCGCCCTGGACCGCTACCTCGCCATCACGTCGCCCTTTCGCTACCAG  601 AGTTTGCTGACGCGCGCGCGAGCGCGGGCCCTCGTGTGCACAGTGTGGGCCATCTCGGCG  661 TTGGTGTCCTTCCTGCCCATCCTCATGCACTGGTGGCGGGCCGAGAGCGACGAAGCGCGC  721 CGCTGCTACAACGACCCCAAGTGCTGCGATTTCGTCACCAACAGGGCCTACGCCATCGCC  781 TCGTCCGTCGTCTCCTTCTACGTGCCCCTGTGCATCATGGCCTTCGTGTACCTGCGGGTG  841 TTCCGCGAGGCCCAAAAACAGGTAAAGAAGATCGACAGCTGCGAGCGCCGCTTCCTCGGC  901 GGCCCAGCCCGGCCGCCCTCGCCTGAGCCCTCGCCGTCACCTGGGCCACCGCGCCCCGCA  961 GACTCGCTGGCCAACGGGCGCTCCAGCAAGCGGCGGCCGTCGCGCCTCGTGGCTCTGCGC 1021 GAGCAGAAGGCGCTCAAGACACTGGGCATCATCATGGGTGTGTTCACGCTCTGCTGGCTG 1081 CCCTTCTTCCTGGCCAACGTGGTGAAGGCTTTCCACCGCGACCTGGTGCCGGATCGCCTC 1141 TTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCATGTACTGC 1201 CGCAGCCCCGACTTCCGCAAGGCTTTCCAGCGCCTGCTCTGCTGCGCGCGCCGGGCCGCC 1261 TGCAGACGCCGCGCAGCCCACGGGGACCGGCCGCGCGCCTCCGGCTGCCTGGCGAGAGCT 1321 GGGCCGCCGCCGTCCCCCGGAGCTCCCTCGGACGACGACGACGACGACGCCGGGACCACC 1381 CCACCGGCGCGCCTGCTGGAGCCCTGGACCGGCTGCAACGGCGGGACAACCACTGTGGAC 1441 AGCGATTCGAGCCTGGACGAGCCGGGGCGCCAGGGCTTCTCCTCGGAGTCCAAGGTGTAG 1501 AGAGCCAGGCTCTCTGGGCGCACGG

5.8.7 Frog β₁-AR

The GenBank™ Accession No. for the Xenopus laevis β ₁-AR Gene is Y09213. The Open Reading Frame extends from nucleotide 301 to nucleotide 1458 (shown in bold) (SEQ ID NO:194)

   1 ACCACCAACGTGGCACCTGCAGCTGAGGGACTAGAAAACTCCATAGCCCAGAGGAGTCAC    61 TGGCAGCCACAACTGTACTGAAAGTGTAGCAGCTCACAAGCCCCCGGCTTATTCATCCAG   121 GAGACAGAGAGACTGGCACAGTCCAGCCCAGTGGCACGAGAGTCTGCACCAACCAGGGGG   181 AGTTATAGTTTCTGGACACAAGAGACTACCTGGCATCCCGCTGGCACCGACACTTTCTTT   241 CTGTTCTGATATTCTGTTGCCCAAATGATCTGAGGCTCCAGGCTAGGACCTATGCCCATC   301 ATGGGAGACGGTTGGGGGCCTATGGAGTGCAGGAACAGGTCTGGTACCCCTACAACAGTG   361 CCCAGCCCTATGCACCCCCTGCCCGAGCTCACTCACCAGTGGACTATGGGAATGACTATG   421 TTCATGGCGGCCATCATCCTCCTCATCGTCATGGGCAACATCATGGTCATTGTGGCCATT   481 GGGAGGAACCAGAGGCTCCAGACCTTGACCAACGTCTTCATCACGTCCTTGGCTTGTGCC   541 GACCTCATTATGGGTTTGTTTGTTGTGCCCCTTGGTGCCACGTTGGTGGTGAGTGGCAGG   601 TGGCTGTACGGGTCGATATTCTGTGAGTTCTGGACGTCAGTGGACGTATTGTGCGTCACG  661 GCGAGTATAGAGACCCTGTGCGTCATCTCCATCGACCGCTACATCGCCATCACCTCACCC  721 TTCCGCTACCAGAGTCTCCTGACCAAGGGCCGTGCCAAGGGAATCGTGTGCAGCGTGTGG  781 GGCATCTCAGCCCTGGTCTCGTTCCTGCCCATCATGATGCACTGGTGGAGGGACACTGGC  841 GACCCCCTGGCCATGAAATGTTACGAGGATCCTGGGTGCTGTGATTTTGTCACCAACAGA  901 GCTTACGCCATCGCCTCGTCCATCATCTCCTTCTATTTCCCACTCATCATCATGATCTTC  961 GTCTACATCAGGGTCTTCAAGGAGGCGCAGAAGCAGATGAAGAAGATTGACAAGTGCGAG 1021 GGCAGGTTCTCCCATAGCCACGTCCTGAGCCACGGCAGGTCCAGCCGGAGGATCCTCTCC 1081 AAAATCCTGGTGGCCAAAGAGCAGAAAGCCTTGAAGACCCTCGGGATTATCATGGGCACC 1141 TTCACCCTGTGCTGGTTGCCCTTCTTCTTGGCCAACGTGGTCAATGTCTTCTACAGGAAC 1201 CTGATCCCAGACAAACTCTTCCTCTTCCTCAACTGGCTGGGCTACGCCAACTCCGCGTTT 1261 AACCCCATCATCTACTGCAGGAGCCCAGACTTCAGGAAGGCTTTCAAGAGACTCCTGTGT 1321 TGCCCCAAAAAGGCAGATCGGCACCTCCACACTACTGGGGAGCTCTCCCGATACTCGGGG 1381 GGCTTTGTTAACTCTTTAGACACCAATGCTTTGGGTATGTGTTCTGAATGTAATGGGGTG 1441 CGGACGTCATTGGACTGAAATTAATTATTTATTGTGGGTCGGAGGGAGATTGAATAAGTG 1501 GGTGCGGGGCCCCAAATAACAGGTAGGTTCCAGGCAACCTCACTGCAGATTCTTGGAATG 1561 TAGAGGGTTCCCCAGGATAGGAGT

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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. ore specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented are as described in the claims below.

204 1 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 1 ccgcgcccat gccga 15 2 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 2 ggccgacgac aggtt 15 3 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 3 atgagcagca cgatg 15 4 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 4 gggcgctcgc cctggcgcct ccgaaccctg caacc 35 5 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 5 atgggcgcgg gggcgctcgc cctggcgcct ccgaa 35 6 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 6 gcctccgaat cggcatgggc gcgggggcgc tcgcc 35 7 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 7 accccccgcg cccggcctcc gaatcggcat gggcg 35 8 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 8 gggtgctcgt cctgggcgcc tccgagcccg gtaac 35 9 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 9 atgggcgcgg gggtgctcgt cctgggcgcc tccga 35 10 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 10 gcagctcggc atgggcgcgg gggtgctcgt cctgg 35 11 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 11 cccggcctcc gcagctcggc atgggcgcgg gggtg 35 12 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 12 ccgcccccgg cctccgcagc tcggcatggg cgcgg 35 13 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 13 accccccgcc cccggcctcc gcagctcggc atggg 35 14 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 14 gggcgctcgc cctgggcgcc tccgaaccct gcaac 35 15 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 15 atgggcgcgg gggcgctcgc cctgggcgcc tccga 35 16 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 16 gcagctcggc atgggcgcgg gggcgctcgc cctgg 35 17 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 17 cccggcctcc gcagctcggc atgggcgcgg gggcg 35 18 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 18 gggcgctcgc cctgggcgcc tccgagccct gcaac 35 19 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 19 atgggcgcgg gggcgctcgc cctgggcgcc tccga 35 20 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 20 gcagctcggc atgggcgcgg gggcgctcgc cctgg 35 21 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 21 cccggcctcc gcagctcggc atgggcgcgg gggcg 35 22 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 22 gcgggggcgt cgccctgggt gcctccgagc cctgc 35 23 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 23 atggggcggg ggcgtcgccc tgggtgcctc cgagc 35 24 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 24 cgcagccggt atggggcggg ggcgtcgccc tgggt 35 25 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 25 cccccgcctc cgcagccggt atggggcggg ggcgt 35 26 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 26 gggcgctcgt cctgggcgcc tccgagcccg gtaac 35 27 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 27 atgggcgcgg gggcgctcgt cctgggcgcc tccga 35 28 35 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 28 gcaactcggc atgggcgcgg gggcgctcgt cctgg 35 29 34 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 29 cagctcggca tgggcgcggg ggtgctcgtc ctgg 34 30 34 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 30 gcagctcggc atgggcgcgg gggtgctcgt cctg 34 31 33 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 31 agctcggcat gggcgcgggg gtgctcgtcc tgg 33 32 33 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 32 gcagctcggc atgggcgcgg gggtgctcgt cct 33 33 32 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 33 gctcggcatg ggcgcggggg tgctcgtcct gg 32 34 32 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 34 gcagctcggc atgggcgcgg gggtgctcgt cc 32 35 31 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 35 ctcggcatgg gcgcgggggt gctcgtcctg g 31 36 31 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 36 gcagctcggc atgggcgcgg gggtgctcgt c 31 37 30 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 37 tcggcatggg cgcgggggtg ctcgtcctgg 30 38 30 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 38 gcagctcggc atgggcgcgg gggtgctcgt 30 39 29 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 39 cggcatgggc gcgggggtgc tcgtcctgg 29 40 29 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 40 gcagctcggc atgggcgcgg gggtgctcg 29 41 28 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 41 ggcatgggcg cgggggtgct cgtcctgg 28 42 28 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 42 gcagctcggc atgggcgcgg gggtgctc 28 43 27 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 43 gcatgggcgc gggggtgctc gtcctgg 27 44 27 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 44 gcagctcggc atgggcgcgg gggtgct 27 45 26 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 45 catgggcgcg ggggtgctcg tcctgg 26 46 26 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 46 gcagctcggc atgggcgcgg gggtgc 26 47 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 47 atgggcgcgg gggtgctcgt cctgg 25 48 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 48 gcagctcggc atgggcgcgg gggtg 25 49 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 49 tgggcgcggg ggtgctcgtc ctgg 24 50 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 50 gcagctcggc atgggcgcgg gggt 24 51 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 51 gggcgcgggg gtgctcgtcc tgg 23 52 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 52 gcagctcggc atgggcgcgg ggg 23 53 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 53 ggcgcggggg tgctcgtcct gg 22 54 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 54 gcagctcggc atgggcgcgg gg 22 55 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 55 gcgcgggggt gctcgtcctg g 21 56 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 56 gcagctcggc atgggcgcgg g 21 57 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 57 cgcgggggtg ctcgtcctgg 20 58 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 58 gcagctcggc atgggcgcgg 20 59 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 59 gcgggggtgc tcgtcctgg 19 60 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 60 gcagctcggc atgggcgcg 19 61 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 61 cgggggtgct cgtcctgg 18 62 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 62 gcagctcggc atgggcgc 18 63 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 63 gggggtgctc gtcctgg 17 64 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 64 gcagctcggc atgggcg 17 65 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 65 ggggtgctcg tcctgg 16 66 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 66 gcagctcggc atgggc 16 67 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 67 gggtgctcgt cctgg 15 68 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 68 gcagctcggc atggg 15 69 14 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 69 ggtgctcgtc ctgg 14 70 14 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 70 gcagctcggc atgg 14 71 14 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 71 ggtgctcgtc ctgg 14 72 13 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 72 gcagctcggc atg 13 73 13 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 73 gtgctcgtcc tgg 13 74 12 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 74 gcagctcggc at 12 75 12 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 75 tgctcgtcct gg 12 76 11 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 76 gcagctcggc a 11 77 11 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 77 gctcgtcctg g 11 78 10 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 78 gcagctcggc 10 79 10 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 79 ctcgtcctgg 10 80 9 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 80 gcagctcgg 9 81 14 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 81 cgcgcccatg ccga 14 82 14 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 82 ccgcgcccat gccg 14 83 13 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 83 gcgcccatgc cga 13 84 13 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 84 ccgcgcccat gcc 13 85 12 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 85 cgcccatgcc ga 12 86 12 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 86 ccgcgcccat gc 12 87 11 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 87 gcccatgccg a 11 88 11 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 88 ccgcgcccat g 11 89 10 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 89 cccatgccga 10 90 10 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 90 ccgcgcccat 10 91 9 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 91 ccatgccga 9 92 9 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 92 ccgcgccca 9 93 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 93 cacccccgcg cccat 15 94 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 94 acccccgcgc ccatg 15 95 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 95 cgcgcccatg ccgag 15 96 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 96 gcgcccatgc cgagc 15 97 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 97 cgcccatgcc gagct 15 98 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 98 gcccatgccg agctg 15 99 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 99 cccatgccga gctgc 15 100 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 100 ccatgccgag ctgcg 15 101 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 101 catgccgagc tgcgg 15 102 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 102 atgccgagct gcgga 15 103 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 103 gcgcccatgc cgagct 16 104 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 104 cgcccatgcc gagctg 16 105 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 105 gcccatgccg agctgc 16 106 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 106 cccatgccga gctgcg 16 107 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 107 ccatgccgag ctgcgg 16 108 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 108 catgccgagc tgcgga 16 109 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 109 atgccgagct gcggag 16 110 16 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 110 tgccgagctg cggagg 16 111 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 111 cgcgcccatg ccgagct 17 112 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 112 gcgcccatgc cgagctg 17 113 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 113 cgcccatgcc gagctgc 17 114 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 114 gcccatgccg agctgcg 17 115 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 115 cccatgccga gctgcgg 17 116 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 116 ccatgccgag ctgcgga 17 117 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 117 catgccgagc tgcggag 17 118 17 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 118 atgccgagct gcggagg 17 119 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 119 ccgcgcccat gccgagct 18 120 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 120 cgcgcccatg ccgagctg 18 121 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 121 gcgcccatgc cgagctgc 18 122 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 122 cgcccatgcc gagctgcg 18 123 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 123 gcccatgccg agctgcgg 18 124 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 124 cccatgccga gctgcgga 18 125 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 125 ccatgccgag ctgcggag 18 126 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 126 catgccgagc tgcggagg 18 127 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 127 acccccgcgc ccatgccga 19 128 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 128 cccgcgccca tgccgagct 19 129 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 129 ccgcgcccat gccgagctg 19 130 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 130 cgcgcccatg ccgagctgc 19 131 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 131 gcgcccatgc cgagctgcg 19 132 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 132 cgcccatgcc gagctgcgg 19 133 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 133 gcccatgccg agctgcgga 19 134 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 134 cccatgccga gctgcggag 19 135 19 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 135 ccatgccgag ctgcggagg 19 136 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 136 cacccccgcg cccatgccga 20 137 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 137 acccccgcgc ccatgccgag 20 138 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 138 cccccgcgcc catgccgagc 20 139 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 139 ccccgcgccc atgccgagct 20 140 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 140 cccgcgccca tgccgagctg 20 141 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 141 ccgcgcccat gccgagctgc 20 142 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 142 cgcgcccatg ccgagctgcg 20 143 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 143 gcgcccatgc cgagctgcgg 20 144 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 144 cgcccatgcc gagctgcgga 20 145 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 145 gcccatgccg agctgcggag 20 146 20 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 146 cccatgccga gctgcggagg 20 147 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 147 cacccccgcg cccatgccga g 21 148 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 148 acccccgcgc ccatgccgag c 21 149 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 149 cccccgcgcc catgccgagc t 21 150 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 150 ccccgcgccc atgccgagct g 21 151 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 151 cccgcgccca tgccgagctg c 21 152 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 152 ccgcgcccat gccgagctgc g 21 153 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 153 cgcgcccatg ccgagctgcg g 21 154 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 154 gcgcccatgc cgagctgcgg a 21 155 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 155 cgcccatgcc gagctgcgga g 21 156 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 156 gcccatgccg agctgcggag g 21 157 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 157 cacccccgcg cccatgccga gc 22 158 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 158 acccccgcgc ccatgccgag ct 22 159 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 159 cccccgcgcc catgccgagc tg 22 160 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 160 ccccgcgccc atgccgagct gc 22 161 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 161 cccgcgccca tgccgagctg cg 22 162 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 162 ccgcgcccat gccgagctgc gg 22 163 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 163 cgcgcccatg ccgagctgcg ga 22 164 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 164 gcgcccatgc cgagctgcgg ag 22 165 22 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 165 cgcccatgcc gagctgcgga gg 22 166 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 166 cacccccgcg cccatgccga gct 23 167 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 167 acccccgcgc ccatgccgag ctg 23 168 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 168 cccccgcgcc catgccgagc tgc 23 169 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 169 ccccgcgccc atgccgagct gcg 23 170 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 170 cccgcgccca tgccgagctg cgg 23 171 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 171 ccgcgcccat gccgagctgc gga 23 172 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 172 cgcgcccatg ccgagctgcg gag 23 173 23 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 173 gcgcccatgc cgagctgcgg agg 23 174 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 174 cacccccgcg cccatgccga gctg 24 175 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 175 acccccgcgc ccatgccgag ctgc 24 176 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 176 cccccgcgcc catgccgagc tgcg 24 177 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 177 ccccgcgccc atgccgagct gcgg 24 178 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 178 cccgcgccca tgccgagctg cgga 24 179 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 179 ccgcgcccat gccgagctgc ggag 24 180 24 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 180 cgcgcccatg ccgagctgcg gagg 24 181 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 181 cacccccgcg cccatgccga gctgc 25 182 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 182 acccccgcgc ccatgccgag ctgcg 25 183 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 183 cccccgcgcc catgccgagc tgcgg 25 184 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 184 ccccgcgccc atgccgagct gcgga 25 185 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 185 cccgcgccca tgccgagctg cggag 25 186 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 186 ccgcgcccat gccgagctgc ggagg 25 187 1723 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 187 tgctacccgc gcccgggctt ctggggtgtt ccccaaccac ggcccagccc tgccacaccc 60 cccgcccccg gcctccgcag ctcggcatgg gcgcgggggt gctcgtcctg ggcgcctccg 120 agcccggtaa cctgtcgtcg gccgcaccgc tccccgacgg cgcggccacc gcggcgcggc 180 tgctggtgcc cgcgtcgccg cccgcctcgt tgctgcctcc cgccagcgaa agccccgagc 240 cgctgtctca gcagtggaca gcgggcatgg gtctgctgat ggcgctcatc gtgctgctca 300 tcgtggcggg caatgtgctg gtgatcgtgg ccatcgccaa gacgccgcgg ctgcagacgc 360 tcaccaacct cttcatcatg tccctggcca gcgccgacct ggtcatgggg ctgctggtgg 420 tgccgttcgg ggccaccatc gtggtgtggg gccgctggga gtacggctcc ttcttctgcg 480 agctgtggac ctcagtggac gtgctgtgcg tgacggccag catcgagacc ctgtgtgtca 540 ttgccctgga ccgctacctc gccatcacct cgcccttccg ctaccagagc ctgctgacgc 600 gcgcgcgggc gcggggcctc gtgtgcaccg tgtgggccat ctcggccctg gtgtccttcc 660 tgcccatcct catgcactgg tggcgggcgg agagcgacga ggcgcgccgc tgctacaacg 720 accccaagtg ctgcgacttc gtcaccaacc gggcctacgc catcgcctcg tccgtagtct 780 ccttctacgt gcccctgtgc atcatggcct tcgtgtacct gcgggtgttc cgcgaggccc 840 agaagcaggt gaagaagatc gacagctgcg agcgccgttt cctcggcggc ccagcgcggc 900 cgccctcgcc ctcgccctcg cccgtccccg cgcccgcgcc gccgcccgga cccccgcgcc 960 ccgccgccgc cgccgccacc gccccgctgg ccaacgggcg tgcgggtaag cggcggccct 1020 cgcgcctcgt ggccctacgc gagcagaagg cgctcaagac gctgggcatc atcatgggcg 1080 tcttcacgct ctgctggctg cccttcttcc tggccaacgt ggtgaaggcc ttccaccgcg 1140 agctggtgcc cgaccgcctc ttcgtcttct tcaactggct gggctacgcc aactcggcct 1200 tcaaccccat catctactgc cgcagccccg acttccgcaa ggccttccag ggactgctct 1260 gctgcgcgcg cagggctgcc cgccggcgcc acgcgaccca cggagaccgg ccgcgcgcct 1320 cgggctgtct ggcccggccc ggacccccgc catcgcccgg ggccgcctcg gacgacgacg 1380 acgacgatgt cgtcggggcc acgccgcccg cgcgcctgct ggagccctgg gccggctgca 1440 acggcggggc ggcggcggac agcgactcga gcctggacga gccgtgccgc cccggcttcg 1500 cctcggaatc caaggtgtag ggcccggcgc ggggcgcgga ctccgggcac ggcttcccag 1560 gggaacgagg agatctgtgt ttacttaaga ccgatagcag gtgaactcga agcccacaat 1620 cctcgtctga atcatccgag gcaaagagaa aagccacgga ccgttgcaca aaaaggaaag 1680 tttgggaagg gatgggagag tggcttgctg atgttccttg ttg 1723 188 1845 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 188 ccgggtgccg ggcccgcggg ctcggcgcgc tcagaaacat gctgaggtcc cggcggctgt 60 tgcagcagcg gcagcggctc cagcagcggc tccagcagcg gctccggcag cggctccagc 120 ggcggcaact ccggcggcag cagcagcggc ggcggcggcg gcggcagcgc acggctcccg 180 cggggaaggc gcccggcgcc catgcctccg gccccgcgcg gcggctgccc tgacccggcc 240 gcgacctccg cgccccacgt cccccggcgt ggtccccggc cacggcccca gcccgccaca 300 ccccccgccc ccggcctccg cagctcggca tgggcgcggg ggcgctggcc ctgggcgcct 360 cggagccctg caacctgtcg tcggccgcgc cgctccccga cggcgcggcc acggcggcga 420 ggctgctggt gcccgcgtcg ccgtccgcct cgccgctggc cccgaccagc gagggccccg 480 cgccgctgtc gcagcagtgg acggcgggca tcgggctgct gatggcgctc atcgtgctgc 540 tcatcgtggc gggcaacgtg ctggtgatcg cggccatcgc caagacgccg cggctgcaga 600 cgctcaccaa cctgttcatc atgtccctgg ccagcgccga cctggtcatg gggctgctgg 660 tggtgccctt cggggccacg atcgtcatgc ggggccgctg ggagtacggc tccttcctgt 720 gcgagctctg gacctcggtg gacgtgctgt gcgtgacggc cagcatcgag accctgtgtg 780 tcatcgcgct ggaccgctac ctggccatca ccgcgccctt ccgctaccag agcctgctga 840 cgcgcgcgcg cgcgcgggcc ctcgtgtgca ccgtgtgggc catctcggcg ctcgtgtcct 900 tcctgcccat cctcatgcac tggtggcggg ccgggggcga cgaggcgcgc cgctgctaca 960 acgaccccaa gtgctgcgac ttcgtcacca accgggccta cgccatcgcc tcgtccgtcg 1020 tctccttcta cgtgcccctg tgcatcatgg ccttcgtgta cctgcgggtg ttccgcgagg 1080 cgcagaagca ggtgaagaag atcgacagct gcgagcgccg cttcctgggc ggccccgcgc 1140 ggccccccgc gcccccgccc gcgcccgcgc ccgcgccccc gcccgcgccc ggctccccgc 1200 gccccgccgc ggccgccccg ctggccaacg ggcgcgtcgg caggcggcgg ccctcgcgcc 1260 tcgtggcgct gcgcgagcag aaggcgctca agacgctggg catcatcatg ggcgtgttca 1320 cgctgtgctg gctgcccttc ttcctggcca acgtggtcaa ggccttccac cgcgacctgg 1380 tgcccgaccg cctcttcgtc ttcttcaact ggctgggcta cgccaactcg gccttcaacc 1440 ccatcatcta ctgccgcagc cccgacttcc gcagggcctt ccagcgcctg ctgtgctgcg 1500 cgcgccgcgc cgcccgcggg agccacgggg ccgccgggga ccctccgcgg gcccggccgc 1560 cgccgtcccc cggggccgcc tcggacgacg acgacgacga cgaggacgac gccggggccg 1620 gggccggggc cgcgccgccc gcgcgcctgc tggagccctg ggccggctgc aacggcgggg 1680 cggcggccga cagcgactcc agcctggacg gcgcgggcag ccccgcgggc gcctcggagt 1740 cccgggtgta ggcgcgcggc ctcccggggg gcgcatcggt gtttactcca gacccagagc 1800 aggtgaaccc cgggcccgcg cacccccntc gcattcatcg aggca 1845 189 4749 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 189 tccagccccc tctttctagc cctctccttc cctcatttcc ccttctcagg ctccccaact 60 ggcagaacta agctgacaat cctaagccag ggatgcagaa acaagtaatt cacccacatc 120 cacccactga tcatcaagtt tgggcctaaa gcaaatttac atgtttggat aaagaaaagt 180 tgggcttccc tagtagctga gacccatctt cagtccttgg atgggggaag atcccctaga 240 gaaggagatg gcaacccact ctagtattct tgcctggaaa atcccatagg cagaggagcc 300 tggtggctac agcccatggg gttgcaagag tcagacacaa cttagctact aaaaccacca 360 cccatggctt atgaatacac attgctgtta gctctcgact tagggagctc tctccaaggt 420 aagaatatga gtttgttcct ttcagaaact attcttttta ttccaatgct agaaggatgt 480 gtgagcatta tgtaacattt tcatgcaccc ttaagtgggt aattagaagc tctttatttc 540 tcaggattca attaaaagct ttttattttc aaggctgagt tgaggaccag tactgtggtg 600 gaattagaca aggggcttgc acacctttgg ctacattgtg tgttgatggg ccaccttcct 660 gtaggtacct ccccacatat agtcacacca ctgcagagct aacgactcac taattttaaa 720 cccattcagt tgccaaccca acagcctttg atataacttt acatgctatt ggattttaat 780 ctttttgagt atttatatat gttttcttct ctcatccctc caaaattaat cctagagttt 840 tgagaatctg ggaacttggg caaaggagaa ggcaacgcag cagaccaaga aatttgaaat 900 ctcagttcac tactgtgtca cccaaagtca atgtaccttt tttgtttgga ccggcccagc 960 tcaagtcata caatcacgtg agtaacagac cacaaaatcc aggtgttatt actgaacatg 1020 acaagtctga aaagtaatta cacgtgttct agcttccgtg gcggtgtcat ttactctaac 1080 atgcctgtcc ttaagcctct ctctctcttt acattaccgg cacacaccgg tgcaccatac 1140 tcacacatcc atcagctggg acctgggagt gtgtattatt ccaactggtc ctcagcatta 1200 gctgtcagat gtcacaaccc ccygccgttt tctgcatctg ctgccccggg aagcgagaag 1260 aagcttgcaa gaatagctcc cgggaacgtt cctgaaagat tggcgctctg ctttagcaag 1320 gcgctcgctg gaaagtttct tctaaccgct cacacccgcc tccgatccga tccccgagct 1380 ggcaggacgc gagctggctg ggactcctct tgacagagga agggctttac acaccaccct 1440 cctaggctgc ccaatacaag aaacagtctt gcagccagac tcctccacac ccagcgaaca 1500 gaccgtccaa ggcgctccgg tgtttcgaga acaccgaagt cccctccctg ctaaagggcg 1560 cgtgagctct gctctgcagg aaacctgggc actggaggta gatgggatgg gtggcggcgg 1620 gtagagccgg ggcgcagcgg aaagcaaacg ccggaggcaa acggggcgca ggagagggga 1680 gattgggtgc cgccgtaggg gccagggtga aagccgggcg cggacgggaa ccgaggggaa 1740 ctgggcactg gagccaagcg ggctctggaa gggacgcgcg ggcaggaacc cgcgagcgct 1800 ggggaggggc ttgcttggcg atctgccccg gactccctag agccgcagaa ccgccggtgg 1860 aggcggggtg ctaggagttg gcggggccgg gtgggggtgg gggggaacca gagaggggcg 1920 tgccttcgcc aggattggct gcaggagcct gacgcgagnn nccgggggtt ggctcggggg 1980 agtgggagcc gggtggggtg ggtgctgggt gccggggctg cgggctccgc gagctcagaa 2040 acatgctgag gtcccggcag ctgttccagc agcgacacca ctccagcagc agccgcggcg 2100 gctgcggcgg cgacaggcac cggctccggc ggggaaggcg cccggcgcca tgcctccggc 2160 cccgcgccgc gctgcgctga cctggccgcg acctccctcc gcgcgccccg ccgttcgggc 2220 ctctgggggg ttccccaacc gcggcccaac tccgccacac ccctctcccc cggcctccgc 2280 agctcggcat gggcgcgggg gcgctcgccc tgggcgcctc cgagccctgc aacctgtcat 2340 tcgccgcgcc ggtccccgac ggcgcggcca cggcggcgcg gctgctagtt cccncgtcgc 2400 cgctccgcct cgctgctgac ctcggccagc gagggacccc gctgctgtcg cagcagtgga 2460 cggtcggcat gggcctgctg atggcattca ttgtgctgct catcgtggcg ggcaacgtgc 2520 tggtgatcgt ggccatcgcc aagactccgc ggctgcagac gctcaccaac ctcttcatca 2580 tgtcgctggc cagcgcagac ctggtcatgg gtctgctggt agtgccgttt ggagccacaa 2640 tcgtggtgtg gggccgctgg gagtatggct ccttcttctg cgagctctgg acctcggtgg 2700 acgtgctgtg cgtgacggcc agcatcgaga ccctgtgtgt catcgccctg gaccgctacc 2760 tcgccatcac gtcgcccttc cgctaccaga gcctgctgac ccgcgcgcga gcgcgggccc 2820 tcgtgtgcac cgtgtgggcc atctcggcgc tggtgtcctt cctgcccatc ttcatgcagt 2880 ggtgggggga caaggacgcc aaggcgagcc ggtgctacaa cgaccccgag tgctgcgact 2940 tcatcatcaa cgagggctac gcgatcacct cttccgtcgt ctccttctac gtgcccctgt 3000 gcatcatggc cttcgtgtac ctgcgggtgt tccgcgaggc ccagaagcag gtgaagaaga 3060 tcgacagctg cgagcgccgc ttcctcagcg gccccgcgcg gctgccctcg cccgcgctct 3120 cgcccggggc gccgctccct gccgccgcgg tggccaacgg gcgcgccaac aagcggcggc 3180 cctcgcgcct cgtggccctg cgcgagcaaa aggccctcaa gacgctgggc atcatcatgg 3240 gcgtgttcac gctctgctgg ctgcccttct tcctggccaa cgtggtgaag gccttccacc 3300 gcgacctggt gcccgaccgc ctcttcgtct tcttcaactg gctgggctac gccaactcgg 3360 ccttcaaccc catcatctac tgccgcagcc ccgacttccg caaggccttc cagcgcctgc 3420 tctgctgcgc gcgccgggcc gcctgcggga gccacggggc cgccggggac ccgccgcgcg 3480 ccgcgggctg cctggcggtg gcccggccgt cgccgtctcc cggggccgcc tcggacgacg 3540 acgacgacga cgacgaagac gacgtcgggg ccgcgccgcc cgtgcgcctg ctgcagccct 3600 gggctggcta caacggcggg gcggcggcga acagcgactc gagcccggac gagccaagcc 3660 gcccgggctg cggctcggaa tccaaggtgt agggacgggc gcccctcccc gccttccccg 3720 gcttccccag tccgggagcg ggctgtgcgc tccaggagca agagaacccg ggcgmccctg 3780 aaccgcttcc ccgggaaaga ggtctgtgtt tactcgagac cgtaaagcag gtgaactcga 3840 agcctgcgaa cctcgtctgc atcatccaag ggcaaatagg aaagccacgg accgtcgcac 3900 agaaaggaaa gtttggggag aggtgggaga agtttgggga ggggtggaag agtggcttac 3960 tgattgttct tggggttctt tttcctgttt ctggtccagc cttctgtgtg tgcgtgtgat 4020 gcatctttag agtccctcct ccccccgccc ccccgcgacg tggcttttaa cactttctgc 4080 gatgactggg aagggaaggg ggaagcgtta ggaggtaaaa gtctctcgac ttagtttcca 4140 tcccattcct gggaacagaa gcggtcagcc agagagagag gagagagaat gacactttat 4200 caggacgttg tttccttttg cttttcagag aaataccatt ttaatttctg aggaattatt 4260 tctcctgttc tgaaagccga gggcaaggat ggatgcaaaa atcgcgtttc aggaagtttt 4320 atgctcttct tggaacaagc ctcaccttgc ttccctttcg gagggcaagc gggctgtccc 4380 tgaacgcctc ctcggtggtc aggctgaggg gttcctacct cactcacacg gtgcacattg 4440 cacggccaga tagagagact tgtttatatt aaacagctta tttatgtatc aatactagtt 4500 ggaaggacca ggcgctgagc cttcgtgaca tgtgactctg tccattgaag acaggacaga 4560 aaaaggaaaa agaaaaaagg aaaacaattc agattactgc acatgtggta tagacaaaaa 4620 atcaaaacaa aaaagccgtg attcaaagtg gcattttttt tgcacagtat taggaactgt 4680 aaagtccaca gaaaatgtta tttgcacaaa aagaaatgaa atatttttta atgggagtgg 4740 ggtggggca 4749 190 2170 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 190 gactccccag agctgctgaa ccgccggggg gagggggtgg gggagttggc gggggggggg 60 ggggagaacc agatcggggc gtgcttcctc ggattgctgc aatagctgac gcgaggcccc 120 gggggttggc tccagggagt gggatgggag cgggtggggt ggtgctgggt gccggggctg 180 cgggttccgg cgctcagaaa ctgctgaggt cgcagctgtc cagcagcgac accgctccag 240 cagcagcggc ggcggcgggg cggcgacgcg cacagcctgg cggggaaggc gccctgcgcc 300 catgcctccg gcccccgccg cggcgccctg accggccgcg acctccctcg tcgcgccccg 360 ccgcccgggc cttggggggg tcccgaccgc gcccaactcc gccacacccc ccgcccccgc 420 ctccgcagcc ggtatggggc gggggcgtcg ccctgggtgc ctccgagccc tgcaacctgt 480 catcggcgcg ccgctcccga cggccggcca ccgcggcgcg gctgctggtg cctgcgtccc 540 ctccgcctcg ctctgacccc accagcgagg gatccgtgca gctgtcgcag cagtggacgg 600 tggcatgggc tcctgatgcg ctcatcgtgc tgctcatcgt ggcgggcaac gtgctgggat 660 cgtggcatcg ccaagcgccg aggctgcaga cgctcaccaa cctcttcatc atgtcctggc 720 caggccgacc tgtcatgggg ctgctggtgg tgccattcgg ggccaccatc gggtgtgggg 780 cgctgggaga cggctccttc ttctgcgagc tctggacgtc ggtggacgac tgtgcgtacg 840 gccagttcga gaccctgtgt gtcatcgccc tggaccgcta cctcgcatca cgtccccttt 900 cgcaccagag cctgctgacc cgcgcggcac gggccctcgt gtcaccgtgt ggccatctcg 960 ccctggtgtc cttcctgccc atcctcatgc actggtggcg gacaaggggc cgaggcagcc 1020 gctgctacaa cgaccccaag tgctgcgatt tcgtcacaac agggctacgc catccctcgt 1080 ccgtggtctc cttctacgtg cccttgtgca tcaggccttc gttacctgcg gtgttccgcg 1140 aggcccagaa gcaggtgaag aagatcgaca ctgcgagcgc gctttctcgc agccccgcgc 1200 ggccgccctc gcccgcgccc tcgcccggtc cccgctcctg ccgccctgcc gcagccccgg 1260 tagccaacgg gcgcaccagc aagagcggcc ctccgcctcg tgccctgcga gagcagaagg 1320 cgctcaagac gctgggcatc acatgggcgt ttcacgctcg ctggctaccc ttcttcctgg 1380 ccaacgtggt gaaggcctcc accgcgactg gtgcccaccg cctcttcgtc ttcttcaact 1440 ggctgggcta cgccactcgg ccttaacccc atctctactg ccgcagccct gacttccgca 1500 aggccttcca gccctgctct gtgcgcgcgc gggtcgcccg cgggagctgc gcggccgccg 1560 gggatgggcg cgcgcctcgg ctgcctgcgg tggcccggcc gccgccgtcg cccggggccg 1620 cctcggcgac gacgagacga agaaacgtcg gggccgcgcc gccggcgccc ctgctggagc 1680 cctggccgga taaacggcgg gcggcacgtg acagcgactc gagcctggac gagcggacgc 1740 cgggggccgg cctcggaacc aaggtgtagg gcccagcgct ccctccccac ctcccagggg 1800 gatgcagctc tgcgcgagag aagagaaccc ggcgccccga aaggcttccc ggggatgagg 1860 agactgtgtt tatcgagacc gaaagcaggt gaactcgcaa acctcgtctg ctcatctaag 1920 caaacagaaa gccggaccgc tgcacagaaa ggaaagtttg gggagggtgg agagtggttg 1980 ctcattttct tgagttcttt tctctgtttg tggtccgtcc tcctttgtgt gtgctgtgat 2040 gcactttaga tttttattcc cccaggtggt ttttaacact tttgcgaaga cagggaaggg 2100 tgggagaagc aggagagttt ttaaaaaggt gtctcaactg gcttccatcc gttcccggga 2160 cggagcagtc 2170 191 3563 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 191 ggtaccagag tacaaacgtc ggtgttaagg tgttggtact ggagtcaaaa gtctgaagcg 60 tcattctctg agttctttta cgcggggtgg gggttggggg tggggcgtgt catttactcc 120 ggacttaatg ttgcccaaac ttctccttta tactcagaac tcatccatcc ttttccttag 180 ctgggattgg gaacgtgtgt gtccaactgg tccttggcgt tggagttaca gccacaaacc 240 ttcccttcct tccccatcca ctgcacttga gggagagaga gacagctctg cggaagaaca 300 gcgcccagag gcctttctga cagatggcac tctgctcccc aatccgcctg gctgcaaagt 360 ttcctctata actaacaccc acttcctatt ccccaagtgt cattctggcc gggtcccctc 420 gcgatcggga agagccaggc tgcctgatgg cagaacagtc tcacagctaa attctctaat 480 cccacagata ggctgtccaa ggcatctccg gaagccagca agtttccctt ctagctacca 540 ggaccatgca ctagaggtag aaaggcgagg ggctacgggg cgccgaaggg aaaccggctc 600 tgcgggaggg caaaatgaga gctgggtgca gggcaagcgg acaccacttg ggcggtgggg 660 tgggcacagg aggccggcgg ggcacccaga gggcgcagca gaccagactc tggaagagcc 720 tgagcaggaa agggcgcgct cgctgagccc gtgggtgccg gctgcgtagt ccaccgcaat 780 ctttggagcc tttcaatcgc ggtggagggg gtgctgggtg ttgggggtgg ggcaccattg 840 ttcgggggcg tgccttggac gcgattggct gcgggagcct gacgcgcggc ccgggggctg 900 gctggggggt agggagcgag tgggggggag gtgctgggtg ttggagcccc ggccccgcgc 960 gctcagaaac atgctgagtc ccggcaactc ttccagcagc gacccgtcca gcagcagcgg 1020 cggcggcggc ggcgggacac ggcttggcta cggaggagaa ggcgccggcg tccatgcctc 1080 cggccccaag ccgggctgcc ctgacctggc cgcgacctcc cccgtccccg cgcgcgcccc 1140 cagccccggc tctggggtgc ttcccaggcg cggcccagtc cgccacaccc cccgcgcccg 1200 gcctccgaat cggcatgggc gcgggggcgc tcgccctggc gcctccgaac cctgcaacct 1260 gtcgtcgccg cgccgctgcc cgacggcgcg gccacccggc acgactgctg gtgctcgcgt 1320 cgcctccgcc tcgctgctgc ctccagccag cgaggctcag cgccgctgtc gcagcagtgg 1380 acccgggtat gggcctactc ctggcgctca tctgctgctc atcgtagtgg gcaacgtgtt 1440 gtgatcgtgg ccatcgccaa gaccccgcgg tgcagacgct caccaacctc ttcatcatgc 1500 cctggccagc gccgatctgg tcatgggatg ctggtggtgc ctttcggggc caccatttgg 1560 tgtggggccg ctgggagtac ggctcctctt ctgtgagctc tggacttcgg tagactgcta 1620 tgtgtgacgg ccagcatcga gacctgtgtg tcatcgccct ggaccgctac ctcccatcac 1680 gtcgcccttt cgctaccaga gctgctgacg cgcgcgcgag cgcgggccct ctgtgcacag 1740 tgtgggccat ctccgcgctg tgtccttcct gcccatcctc atgcactggg gcgggccgag 1800 agcgacgaag cgcgccgcgc tacaacgacc ccaagtgctg cgatttctca ccaaccgggc 1860 ctacgccatc gcctcgccgt cgtctccttc tacgtgcccc tgtgctcatg gccttcgtgt 1920 acctccgggt gttcgcgagg cccagaaaca ggtgaagaag atcacagctg cgagcgccgc 1980 ttcctcagcg gccgccccgg ccgccctcgc ccgcgccctc gcatcaccag ggccaccgcg 2040 ccccgcagac cgctggccaa cgggcgctcc agcaagcggg gccgtcgcgc ctcgtggctc 2100 tgcgagagag aaggcgctca agacactggg catcatctgg gtgtgttcac gctctgctgg 2160 ctgccctctt cctggccaac gtggtgaaag ctttcaccgc gacctggtgc cggatcgcct 2220 cttctcttct tcaactggct gggctacgcc aaccggcctt caaccccatc atctactgcc 2280 gcgccccgac ttccgcaagg ctttccagcg ctgctttgct gcgcgcgccg ggccgcctgc 2340 gacgccgcgc agcccacggg gaccggccgg cgcctcgggc tgcctggcga gagctgggcg 2400 ccgccgtccc ccggggctcc ttcggacacg acgacgatga cgccggggcc accccaccgc 2460 gcgcctgttg gagccctggg ccggcgcaac ggcgggacga ccactgtgga cagcattcga 2520 gcctggacga gccgggacgc caggcttctc ctccgagtcc aaggtgtaga ggccaggctc 2580 tccgggcgca cggacgccgc tcccatagtc ccgggctgga cacgggctct catccctaga 2640 ggaagagagc ccgcctgggc cctgagccgc tccccagggg agagaggaat ttctgtttac 2700 tcaagaccga aagcagggaa tgcgaagccc acagatcttt tgaatctccg agacgtacag 2760 aaaagccccg gaccggcgtt gcgcaaaaag gaaagtttgg gagtgttggg agagtgtggc 2820 ttagtgtggc ttatggcttg tcttgagttc ctttctctct gagttcggcc tttcgtgtgt 2880 ttaatgcacc ttaggcaccc cccccccgtg ggttttgaca tttctgcaag gacccgagtg 2940 gaaactaggg gggaggggaa gggggagggt ggagtctaca cctgccttct acttcacacc 3000 taggaactaa gtgttcagct ctggtttggg gtgggccagg agcggtgata attagccagg 3060 aagtgtcctt ttgctttcta aagaaattgc atataattcc ggagtattgg tgtctcctta 3120 aagaaagggg ggaaaggtgg ctgagaaaca gacaatctgg tttcgagaaa ctatttgtgg 3180 acacggttca ccttgctttc tcctggaggg aaaccctgtc cctgcgcgcc tcggtggtct 3240 gctgtgggtc ctctacctca ctctgtgcta ttgcacagca agatagaaag acttgttata 3300 ttaaacagct tatttatgta tcaatatagt tggaaggacc aggcgctgag tctcttctgt 3360 gacatgtgat tctgtcaact aaagtaggac agaacaaaag gaaacagttg ggatattgca 3420 catgtggcta aaaacaaaga tgcaaaaaaa aaaggcagtg gttgaaaggc cttttgcgca 3480 gtgttagaca ttgtaaaatc atagaagttg ttaacttgca caaaaaatta atatttttaa 3540 tgggacgggg aggtgggcga tct 3563 192 4401 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 192 ccaaggaatc tgaaatcgca gttcatgacg tcagcataaa ccgacagtac ctttttgttt 60 gtattgaccc agctcaaatt ataaaatcac atgagtaata aaacacaaaa tacaggtgtt 120 gtcactgaaa agcctaaaat gtaactaaac gcgttttccc ccctcgcggt ggtatcattt 180 actctgtatc tcaacgtttc tgtctcccta agactctccc tttatatcgt gagcacacat 240 ttttgcatca tgctcacaca ttcattagct aggactgagg agtgtgtacg attccaacgg 300 gccctcagcg ttagctgtta gatgcacaaa ccttcacttc ctttccacat ctactgcact 360 tgaggttcaa cagaggatcc ttgcaagaac agtgcccaga gagcattctt gacagatgcg 420 cgcttagctc cagcaacccg ctctgctggg aagtttcttc taaccactaa cacccacctc 480 caatccccca agctgtcacg acgcaggctg gctgggtccc gtcttgacgg gggaagggtt 540 ttacacacat cctgctaggc tgccccacat cacaaccaag ctcgcagggc aaactcctcc 600 aagcctggcg gacaagctgt cccaggcgct ctggcgcttc ctgaacacca aggtcccctc 660 cgcgctcaag ggagctagcg cactgttacg cagggaaccc cggcactgca ggtcgagggg 720 atgccgaggg agcgggggcg cagcaggcag ccgactgctg taggcaaacg gcgcgcagga 780 ggcaaacgag gcgcaggagc cggtgcgaga gcgagtgggc gctgagagag ggggcggggc 840 cccagggggg aggcgagcgc gggagggggc acgggagaac agggaccagg aaccagcggg 900 cgcaggaagg ggtgcgtccg caagaacccg cgggcgcacg ggaggcacta gctccacgat 960 cagctcggga ctctcaggag ccgctcaatt gccaacggga gggggctgcg gggagttgga 1020 ggttgggggg gctgaccaga cgggggcgtg cctttgcccg gattggctgc aggaggctga 1080 cgcgaggccc cgggggttgg cttggggagt gggagcgggg tggggtgggt gctgggtgcc 1140 ggagctgcgg gcccggcgcg ctcagaaaca tgctgaggtc ccggcggctc ttccagcagc 1200 ggcagcggct ccagcagcag cggcggcggc ggcggcggcg gcagcgacag cgctcggctc 1260 cggcgggaaa ggcgcccggc gcccatgcct ccggccccgg gccgcggctg ccctgacccg 1320 gccgcgacct ccctctgcgc accccgccgt ccaggcttct ggggtgttcc ccaaccaagg 1380 cccagccctg ccacaccccc cgcctccggc ctccgcaact cggcatgggc gcgggggcgc 1440 tcgtcctggg cgcctccgag cccggtaacc tgtcgtcggc cgcaccgctc cccgacggcg 1500 tggccaccgc ggcgcggctg ctggtgcccg cgtcgccgcc cgcctcgttg ctgcctcccg 1560 ccagcgaagg ccccgagccg ctgtcgcagc agtggacggc gggcatgggt ctgctgatgg 1620 cgctcatcgt gctgctcatc gtggcgggca acgtgctggt gatcgtggcc atcgccaaga 1680 cgccgcggct gcagacgctc accaacctct tcatcatgtc cctggccagc gccgacctgg 1740 tcatggggct gctggtggtg ccgttcgggg ccaccatcgt ggtgtggggc cgctgggagt 1800 acggctcctt cttctgcgag ctgtggacct cggtggacgt gctgtgcgtg acggccagca 1860 tcgagaccct gtgtgtcatc gccctggacc gctacctcgc catcacctcg cccttccgct 1920 accagagcct gctgacgcgc gcgcgggcgc ggggcctcgt gtgcaccgtg tgggccatct 1980 cagccctggt gtccttcctg cccatcctca tgcattggtg gcgggcggag agcgacgagg 2040 cgcgccgctg ctacaacgac cccaagtgct gcgatttcgt caccaaccgg gcctacgcca 2100 tcgcctcgtc cgtggtctcc ttctacgtgc ccctgtgcat catggccttc gtgtacctgc 2160 gggtgttccg cgaggcccag aagcaggtga agaagatcga cagctgcgag cgccgtttcc 2220 tcggcggccc cgcgcggccg ccctcgccct cgccctcgcc ctcgccctcg ccggtccccg 2280 cgccgccgcc cggacctccg cgccccgccg ccgccgctgc caccaccgcc ccgctggtca 2340 acggacgtgc gggtaagcgg cggccctcgc gcctcgtggc cctgcgcgag cagaaggcgc 2400 tcaagacgct gggcatcatc atgggcgtgt tcacgctctg ttggctgccc ttcttcctgg 2460 ccaacgtggt gaaggccttc caccgcgagc tggtgcccga ccgcctcttc gtcttcttca 2520 actggctggg ctacgccaac tcggccttca accccatcat ctactgccgc agccccgact 2580 tccgcaacgc cttccagcga ctgctctgct gcgcgcgcag ggctgcccgc cggcgccacg 2640 cggcccacgg agaccggccg cgcgcctcgg gctgtctggc ccggcccgga cccccgccgt 2700 cgcccggggc cgcctcggac gacgacgacg acgatgtcgt cggggccacg cagcccgcgc 2760 gcctgctgga gccctgggcc ggctgcaacg gcggggcggc ggcggacagc gactcgagcc 2820 tggacgagcc gtgccgcccc ggattcgcct cggagtccaa ggtgtagggc ccggcgccgg 2880 gcgaggacgc cgggcacccc gggaggagga gagcccgggc gccccggaac gacttcccgg 2940 gggaacgagg agatctgtgt ttactcaaga ccgaaagcag gtgaactcga agcccacaat 3000 cctcgtctga atcatccgag gcaaacagaa aagccacgga ccattgcaca aaaaggaaag 3060 tttggggagg gatgggagag tggcttgctg atgttccttg gttttttttc tttctttctt 3120 cctttttttt tttttttttc tgtttgtggt ccggccttct tttgtatgtg cgtgtgatgc 3180 atctttagat tttttttccc ccacgttggt ttttgacact ctctgcgagg accggagtgg 3240 aagttgggtg ggttacggga aggaagaagc attaggaggg gattaaaatc gatcagcgtg 3300 gctcctatcc ctttcccagg aacaggagca gtctaccagc cagagggagg agaatgacag 3360 tttgtcaaga cgtatttctt ttgctttcca gataaatttc attttaattt ctaagtaatg 3420 agttctgctg tcatgaaagc aaagagaaag gatggaggca aaaaaaaaaa aattcacgtt 3480 tcaagaaatg ttaagctctt cttggaacaa gccccacctt gctttccttg tgtagggcaa 3540 accggccgtc ccccgcgcgc ctgggtggtc aggctgaggg atttctacct cacactgtgc 3600 gtttgcacag cagatagaac gacttgttta tattaaacag cttatttatg tatcaatatt 3660 agttggaagg accaggcgca gagcctctct ctgtgacatg tgactctgtc aattgaagac 3720 aggacattag agagagagaa acagttcaga ttactgcaca tgtggataaa aacaaaaaca 3780 acaaaaaaag gagtggttca aaatgccatt tttgcacagt gttaggaatt acaaagtcca 3840 caggagatgt tacttgcaca aaaagaaatt aaatattttt taaaggggga ggggctgggc 3900 agatcttaaa aactaaaata aaattcaaac tctacttctg ttgtctagta tgttattgag 3960 ctaatgattt attggggaaa ataccttttt atactccttt atcatggtac tgtaactgta 4020 tccatattat aaatataatt atcttaagga ttttttattt tttttttttt atgtccaagt 4080 gcccacgtga atttgctggt gaaagttagc acttgtgtgt gaactctact tcctcttgtg 4140 tgttttacca agtatttata ctctggtgca actaactact gtgtgaggaa ttggtccatg 4200 tgcaataaat accaatgaag cacaatcaag attatgtact gtgtgtctgt aaagggtcag 4260 tgataatgaa aaagacagtt tgttttgttc aaaatataga ctggatttcc catagagctc 4320 ttttaataga ctttcatgac tcaataacat agcaaaatgc ctccagacct aaataaggtg 4380 tttacctact gagagctgca g 4401 193 1525 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 193 gacctccccg cgcgggcccc gcagccccgg ctcctggggt gctccccagg cgcggcccag 60 ccccgccaca ccccccgccc ccggcctccg cagctcggca tgggcgcggg ggcgctcgcc 120 ctgggcgcct ccgaaccctg caacctgtcg tccgccgcgc cgctgcccga cggtgcggcc 180 accgcggcgc ggctgctggt gctcgcgtcg cctcccgcct cgctgctgcc tccagccagc 240 gagggctcag cgccgctgtc gcagcagtgg accgcgggta tgggcctact cgtggcgctc 300 atcgttctgc tcatcgtggt gggtaacgtg ctggtgatcg tggccatcgc caagaccccg 360 cggctgcaga cgctcaccaa cctcttcatc atgtccctgg ccagcgctga tctggtcatg 420 ggattgctgg tggtgccttt cggggccacc atcgtggtgt ggggccgctg ggagtacggc 480 tccttcttct gcgagctctg gacttcggta gatgtgctgt gtgtgacggc cagcattgag 540 accctgtgtg tcatcgccct ggaccgctac ctcgccatca cgtcgccctt tcgctaccag 600 agtttgctga cgcgcgcgcg agcgcgggcc ctcgtgtgca cagtgtgggc catctcggcg 660 ttggtgtcct tcctgcccat cctcatgcac tggtggcggg ccgagagcga cgaagcgcgc 720 cgctgctaca acgaccccaa gtgctgcgat ttcgtcacca acagggccta cgccatcgcc 780 tcgtccgtcg tctccttcta cgtgcccctg tgcatcatgg ccttcgtgta cctgcgggtg 840 ttccgcgagg cccaaaaaca ggtaaagaag atcgacagct gcgagcgccg cttcctcggc 900 ggcccagccc ggccgccctc gcctgagccc tcgccgtcac ctgggccacc gcgccccgca 960 gactcgctgg ccaacgggcg ctccagcaag cggcggccgt cgcgcctcgt ggctctgcgc 1020 gagcagaagg cgctcaagac actgggcatc atcatgggtg tgttcacgct ctgctggctg 1080 cccttcttcc tggccaacgt ggtgaaggct ttccaccgcg acctggtgcc ggatcgcctc 1140 ttcgtcttct tcaactggct gggctacgcc aactcggcct tcaaccccat catctactgc 1200 cgcagccccg acttccgcaa ggctttccag cgcctgctct gctgcgcgcg ccgggccgcc 1260 tgcagacgcc gcgcagccca cggggaccgg ccgcgcgcct ccggctgcct ggcgagagct 1320 gggccgccgc cgtcccccgg agctccctcg gacgacgacg acgacgacgc cgggaccacc 1380 ccaccggcgc gcctgctgga gccctggacc ggctgcaacg gcgggacaac cactgtggac 1440 agcgattcga gcctggacga gccggggcgc cagggcttct cctcggagtc caaggtgtag 1500 agagccaggc tctctgggcg cacgg 1525 194 1584 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 194 accaccaacg tggcacctgc agctgaggga ctagaaaact ccatagccca gaggagtcac 60 tggcagccac aactgtactg aaagtgtagc agctcacaag cccccggctt attcatccag 120 gagacagaga gactggcaca gtccagccca gtggcacgag agtctgcacc aaccaggggg 180 agttatagtt tctggacaca agagactacc tggcatcccg ctggcaccga cactttcttt 240 ctgttctgat attctgttgc ccaaatgatc tgaggctcca ggctaggacc tatgcccatc 300 atgggagacg gttgggggcc tatggagtgc aggaacaggt ctggtacccc tacaacagtg 360 cccagcccta tgcaccccct gcccgagctc actcaccagt ggactatggg aatgactatg 420 ttcatggcgg ccatcatcct cctcatcgtc atgggcaaca tcatggtcat tgtggccatt 480 gggaggaacc agaggctcca gaccttgacc aacgtcttca tcacgtcctt ggcttgtgcc 540 gacctcatta tgggtttgtt tgttgtgccc cttggtgcca cgttggtggt gagtggcagg 600 tggctgtacg ggtcgatatt ctgtgagttc tggacgtcag tggacgtatt gtgcgtcacg 660 gcgagtatag agaccctgtg cgtcatctcc atcgaccgct acatcgccat cacctcaccc 720 ttccgctacc agagtctcct gaccaagggc cgtgccaagg gaatcgtgtg cagcgtgtgg 780 ggcatctcag ccctggtctc gttcctgccc atcatgatgc actggtggag ggacactggc 840 gaccccctgg ccatgaaatg ttacgaggat cctgggtgct gtgattttgt caccaacaga 900 gcttacgcca tcgcctcgtc catcatctcc ttctatttcc cactcatcat catgatcttc 960 gtctacatca gggtcttcaa ggaggcgcag aagcagatga agaagattga caagtgcgag 1020 ggcaggttct cccatagcca cgtcctgagc cacggcaggt ccagccggag gatcctctcc 1080 aaaatcctgg tggccaaaga gcagaaagcc ttgaagaccc tcgggattat catgggcacc 1140 ttcaccctgt gctggttgcc cttcttcttg gccaacgtgg tcaatgtctt ctacaggaac 1200 ctgatcccag acaaactctt cctcttcctc aactggctgg gctacgccaa ctccgcgttt 1260 aaccccatca tctactgcag gagcccagac ttcaggaagg ctttcaagag actcctgtgt 1320 tgccccaaaa aggcagatcg gcacctccac actactgggg agctctcccg atactcgggg 1380 ggctttgtta actctttaga caccaatgct ttgggtatgt gttctgaatg taatggggtg 1440 cggacgtcat tggactgaaa ttaattattt attgtgggtc ggagggagat tgaataagtg 1500 ggtgcggggc cccaaataac aggtaggttc caggcaacct cactgcagat tcttggaatg 1560 tagagggttc cccaggatag gagt 1584 195 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 195 ccgcgcccat gccga 15 196 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 196 agccgtaccc gcgcc 15 197 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 197 ccgcgcccat gccga 15 198 15 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 198 agccgtaccc gcgcc 15 199 18 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 199 ctccgaagct cggcatgg 18 200 21 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 200 gcacgtctac cgaagtccag a 21 201 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 201 aggcagtgac cctcaacatt accag 25 202 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 202 ccagtatgca caggtcatcg ttcct 25 203 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 203 atcaaatggg gtgatgctgg tgctg 25 204 25 DNA UNKNOWN SYNTHETIC OLIGONUCLEOTIDE 204 caggtttctc caggcggcat gtcag 25 

What is claimed is:
 1. An isolated antisense oligonucleotide at least 9 to about 35 nucleotides in length, wherein said oligonucleotide specifically binds to a portion of an mRNA expressed from a gene encoding a mammalian β₁-adrenoceptor polypeptide, and further wherein binding of said oligonucleotide to said mRNA is effective in altering the translation of said mRNA into said β₁-adrenoceptor polypeptide in a host cell expressing said mRNA.
 2. The oligonucleotide of claim 1, comprising deoxyribonucleic acid, ribonucleic acid, or peptide-nucleic acid.
 3. The oligonucleotide of claim 2, comprising a derivatized deoxyribonucleic acid, ribonucleic acid, or peptide-nucleic acid.
 4. The oligonucleotide of claim 3, comprising a phosphorothioated deoxyribonucleic acid, ribonucleic acid, or peptide-nucleic acid derivative.
 5. The oligonucleotide of claim 1, comprising a sequence of at least nine contiguous bases from any one of SEQ ID NO:4 through SEQ ID NO:186.
 6. The oligonucleotide of claim 5, comprising a sequence of at least ten contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:93 through SEQ ID NO:186.
 7. The oligonucleotide of claim 6, comprising a sequence of at least thirteen contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:93 through SEQ ID NO:186.
 8. The oligonucleotide of claim 7, comprising a sequence of at least sixteen contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:111 through SEQ ID NO:186.
 9. The oligonucleotide of claim 8, comprising a sequence of at least eighteen contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:127 through SEQ ID NO:186.
 10. The oligonucleotide of claim 9, comprising a sequence of at least twenty contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:147 through SEQ ID NO:186.
 11. The oligonucleotide of claim 10, comprising a sequence of at least twenty-two contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or any one of SEQ ID NO:166 through SEQ ID NO:186.
 12. The oligonucleotide of claim 11, comprising a sequence of at least twenty-four contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185 or SEQ ID NO:186.
 13. The oligonucleotide of claim 12, comprising a sequence of at least twenty-seven contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
 14. The oligonucleotide of claim 13, comprising a sequence of at least thirty contiguous bases from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
 15. The oligonucleotide of claim 14, comprising the sequence of any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.
 16. The oligonucleotide of claim 14, comprising the sequence of any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.
 17. The oligonucleotide of claim 14, comprising the sequence of any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
 18. The oligonucleotide of claim 1, wherein said oligonucleotide is at least 90% complementary to the portion of the mRNA sequence to which it specifically binds.
 19. The oligonucleotide of claim 18, wherein said oligonucleotide is at least 95% complementary to the portion of the mRNA sequence to which it specifically binds.
 20. The oligonucleotide of claim 19, wherein said oligonucleotide is at least 98% complementary to the portion of the mRNA sequence to which it specifically binds.
 21. The oligonucleotide of claim 1, wherein said oligonucleotide has no more than one nucleotide for every nine contiguous nucleotides that is non-complementary to the portion of the mRNA sequence to which it specifically binds.
 22. The oligonucleotide of claim 21, wherein said oligonucleotide has no more than one nucleotide for every ten contiguous nucleotides that is non-complementary to the portion of the mRNA sequence to which it specifically binds.
 23. The oligonucleotide of claim 22, wherein said oligonucleotide has no more than one nucleotide for every eleven contiguous nucleotides that is non-complementary to the portion of the mRNA sequence to which it specifically binds.
 24. The oligonucleotide of claim 23, wherein said oligonucleotide has no more than one nucleotide for every twelve contiguous nucleotides that is non-complementary to the portion of the mRNA sequence to which it specifically binds.
 25. The oligonucleotide of claim 1, wherein said host cell is a mammalian host cell.
 26. The oligonucleotide of claim 25, wherein said host cell is a human cell.
 27. The oligonucleotide of claim 1, wherein said host cell is comprised within a human.
 28. The oligonucleotide of claim 1, wherein said oligonucleotide is at least 9 to about 30 bases in length.
 29. The oligonucleotide of claim 28, wherein said oligonucleotide is at least 9 to about 25 bases in length.
 30. The oligonucleotide of claim 29, wherein said oligonucleotide is at least 9 to about 20 bases in length.
 31. The oligonucleotide of claim 30, wherein said oligonucleotide is at least 9 to about 15 bases in length.
 32. The oligonucleotide of claim 31, wherein said oligonucleotide is at least 9 to about 12 bases in length.
 33. The oligonucleotide of claim 1, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:29 to SEQ ID NO:186.
 34. The oligonucleotide of claim 33, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:93 to SEQ ID NO:186.
 35. The oligonucleotide of claim 34, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:93 to SEQ ID NO:166.
 36. The oligonucleotide of claim 35, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:93 to SEQ ID NO:146.
 37. The oligonucleotide of claim 36, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:93 to SEQ ID NO:126.
 38. The oligonucleotide of claim 36, wherein said oligonucleotide comprises the sequence of any one of SEQ ID NO:96 to SEQ ID NO:112.
 39. A recombinant vector comprising a nucleic acid which expresses (a) the isolated oligonucleotide of claim 1, or (b) an isolated antisense polynucleotide that consists essentially of a sequence that is complementary to the sequence of from about position 87 to position 1520 of SEQ ID NO:187.
 40. The recombinant vector of claim 39, wherein said vector is a viral, retroviral, adenoviral, or adenoassociated viral vector.
 41. A host cell comprising the oligonucleotide of claim 1 or the recombinant vector of claim
 39. 42. A composition comprising the oligonucleotide of claim
 1. 43. A kit comprising the oligonucleotide of claim 1 or the composition of claim
 42. 44. The composition of claim 42, further comprising a pharmaceutical excipient.
 45. The composition of claim 44, further comprising a liposome, a lipid particle, a lipid vesicle, a nanoparticle, a microparticle, a nanocapsule, a nanosphere, or a sphingosome.
 46. The composition of claim 42, further comprising at least a second oligonucleotide of at least 9 to about 35 nucleotides in length, wherein said second oligonucleotide also specifically binds to a portion of an mRNA that encodes a β₁-adrenoceptor polypeptide, and further wherein the nucleotide sequence of said second oligonucleotide is different from the nucleotide sequence of the first oligonucleotide in said composition.
 47. The composition of claim 46, wherein said second oligonucleotide is any one of SEQ ID NO:4 through SEQ ID NO:186.
 48. The composition of claim 42, further comprising at least a first anti-hypertensive agent.
 49. The composition of claim 48, wherein said anti-hypertensive agent is selected from the group consisting of captopril, enalapril, ramipril, cilazapril, fosinopril, and lisinopril.
 50. The composition of claim 42, further comprising at least a second antisense oligonucleotide specific for a mammalian transcription factor mRNA, a renin-specific mRNA, an angiotensin-specific mRNA, an angiotensinogen-specific mRNA, an AT-1 receptor-specific mRNA, or an angiotensin converting enzyme.
 51. The composition of claim 50, wherein said second antisense oligonucleotide is specific for a mammalian angiotensin converting enzyme mRNA.
 52. A method for reducing the level of β₁-adrenoceptor polypeptide in a mammalian host cell, said method comprising providing to said cell, an amount of the oligonucleotide of claim 1, or the composition of claim 42, and for a time effective to reduce the level of said polypeptide in said host cell.
 53. A method for reducing the level of transcription of a β₁-adrenoceptor polypeptide-specific mRNA in a mammalian host cell, said method comprising providing to said cell, an amount of the oligonucleotide of claim 1, or the composition of claim 42, and for a time effective to reduce the level of transcription of said mRNA in said host cell.
 54. The method of claim 52 or 53, wherein said host cell is comprised within a mammal.
 55. The method of claim 54, wherein said mammal is a human.
 56. A method for decreasing hypertension in a mammal, said method comprising administering to said mammal, an amount of the oligonucleotide of claim 1 or the composition of claim 42, and for a time effective to decrease hypertension in said mammal.
 57. A method for reducing the symptoms of a disease or disorder associated with an elevated β₁-adrenoceptor polypeptide level in a mammal, said method comprising administering to said mammal a therapeutically effective amount of the oligonucleotide of claim 1, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or the composition of claim 42, for a time sufficient to decrease the level of said polypeptide in said mammal, thereby reducing the symptoms of said disease or disorder in said mammal.
 58. A method for treating myocardial ischemia or cardiac hypertrophy in an affected mammal, said method comprising administering to said mammal, a therapeutically effective amount of the oligonucleotide of claim 1, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or the composition of claim 42, for a time sufficient to treat said ischemia or said hypertrophy in said mammal.
 59. A method for reducing the level of β₁-adrenoceptor polypeptide in a mammalian host cell, said method comprising expressing in said host cell, a selected genetic construct that comprises a full-length, or substantially full-length β₁-adrenoceptor antisense polynucleotide operably linked to a promoter capable of expressing said polynucleotide in said cell.
 60. The recombinant vector of claim 39, comprising the isolated oligonucleotide of claim
 1. 61. The recombinant vector of claim 39, comprising an isolated antisense polynucleotide that consists essentially of a sequence that is complementary to the sequence of from about position 87 to position 1520 of SEQ ID NO:187.
 62. The recombinant vector of claim 39, comprising an isolated antisense polynucleotide that consists of a sequence that is complementary to the sequence of from about position 87 to position 1520 of SEQ ID NO:187.
 63. The method of claim 52, wherein said method comprises providing to said cell, an amount of the composition of claim 42, and for a time effective to reduce the level of said polypeptide in said host cell.
 64. The method of claim 53, wherein said method comprises providing to said cell, an amount of the composition of claim 42, and for a time effective to reduce said level of transcription of said mRNA in said host cell.
 65. The method of claim 55, wherein said human has, is suspected of having, or is at risk for developing, hypertension, myocardial ischemia or cardiac hypertrophy. 